JP2002531053A - Methods and reagents for analyzing nucleotide sequences of nucleic acids - Google Patents

Abstract

  (57) [Summary] Methods and reagents have been disclosed that are more sensitive, more accurate, and satisfy the need for higher processing resolution of target nucleic acid sequences. The methods and reagents may generally be applied generally to any target nucleic acid sequence and do not require prior information about the presence, location, or identity of the mutation in the target sequence. The reagents of the present invention are mixtures of natural and mass-modified oligonucleotide precursors with high levels of coverage and mass number complexity. A mixture of natural and altered mass oligonucleotide precursors and mass spectrometry analysis using chemical or enzymatic analysis, generally changing the mass of the oligonucleotide precursor prior to analysis by MALDI-TOF A method for analyzing a target nucleic acid sequence is disclosed. The enzymatic assay may be a polymerase extension assay or a ligase assay. A method for performing the method of the present invention is also disclosed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION

The present invention relates to methods and reagents for analyzing the nucleotide sequence of a nucleic acid by mass spectrometry, in particular oligos with high levels of mass number complexity and sequence coverage complexity. The present invention relates to a method for analyzing a nucleotide sequence using a reagent which is a mixture of nucleotide precursors.

[0002]

BACKGROUND OF THE INVENTION

Determining the nucleotide sequence of nucleic acids (DNA and RNA) is extremely important in understanding the function and control of genes and understanding, for example, the relationship between genes and disease management. Analysis of genetic information plays a decisive role in biological experiments. This is especially true for studies that understand the basic genetic and environmental factors associated with disease and the effects of therapeutic potential on cells. With this paradigm shift, in the life science industry,
In order to analyze genetic material obtained from various biological resources, more sensitive,
There is a growing need for more accurate, higher throughput technologies.

Determining the sequence of large numbers of nucleic acids in each human cell is a time-consuming process, and there is an urgent need for rapid, high-throughput analysis that does not sacrifice sensitivity and accuracy. I have. To determine the sequence of the nucleic acid, among others,
Numerous techniques have been developed, including electrophoresis, enzymatic and chemical analysis, array techniques and mass spectrometry.

(Electrophoresis Technology) The slab or capillary polyacrylamide electrophoresis technology used in an automatic DNA sequencer is capable of enhancing novel sequence information on a relatively long (500 to 700 residues or base pairs) DNA fragment. Provide accurate accuracy. Electrophoresis-based techniques provide a large amount of information per sample, but sample preparation and preparation for electrophoresis are time consuming and therefore have limited throughput.

[0005] Enzymatic and chemical analysis There are numerous enzymatic and chemical techniques for determining novel nucleotide sequences of nucleic acids. However, each technology has its own limitations. For example, Maxam and Gilbert [Proc. Natl. Acad. Sci. USA 74: 5460 (1977)] revealed a chemical degradation method and described Sanger et al. [Proc. Natl. Acad. Sci. USA 74: 5463.
(1977)] clarified a chain termination method using primer extension of a complementary strand. Each such technique uses four separate reaction mixtures to create a set of nested fragments that differ by one nucleotide in length and represent the complete nucleotide sequence. By resolving the fragments based on their size and terminal nucleotides, the order of the fragments is determined, thereby determining the nucleotide sequence.

[0006] Single stranded conformation polymorphism (SSCP)
The analysis helps to detect relatively small differences between similar sequences. The technique is simple and could be diversified when combined with multiple dye detection or mass tagging, thereby improving throughput. However, denaturing gradient gel electrophoresis (DGGE), chemical cleavage (CCM), enzymatic cleavage of mismatches (using cleavage), and denaturing high performance liquid chromatography (DHPLC)
The techniques, such as those for detecting heteroduplexes, such as, for example, only indicate whether the mutation is in the target nucleic acid, and are the lowest for identifying and locating the mutation. Only information can be obtained.

[0007] Other techniques, such as those using ligase and polymerase extension assays, help determine if there are mutations at limited positions in the otherwise known target nucleic acid sequence. For example, U.S. Pat. No. 4,988,617 describes a restriction in nucleic acid sequences known separately by measuring the ligation of two natural oligonucleotides designed to hybridize adjacent ones according to a target sequence. Disclosed are methods for determining whether there is a mutation at a given position. U.S. Pat. No. 5,494,810 discloses the use of only natural nucleic acids and the substitution, deletion of specific nucleotides in a known nucleic acid sequence using thermostable ligase and ligase chain reaction (LCR). Disclosed are methods for detecting loss, insertion and transposition. U.S. Patent No. 5,403,709 discloses a method of determining nucleotide sequence using another oligonucleotide as an extension and a third bridging oligonucleotide supporting the first two together for ligation. And WO97 / 35
033 discloses a method for determining the identity of a defined primer at nucleotide 3 'using a polymerase extension assay. Assays may be performed at relatively high throughput, but because they are sequence-specific, a separate set of reagents is required for each target to be analyzed.

[0008] US Patent Nos. 5,521,065, 4,883,750, and 5,2
No. 42,794 (Whiteley et al.) Discloses a method for examining the presence or absence of a target sequence in a mixture of single-stranded nucleic acid fragments. The method involves the reaction of a first probe complementary to the first region of the target sequence and a mixture of the second probe complementary to the second region of the target sequence and a single-stranded nucleic acid fragment. I do. The first and second target regions are adjacent to each other. Hybridization conditions are used so that the two probes can stably hybridize to the relevant target region. Following hybridization, after any of the first and second probes hybridized to the adjacent first and second target regions, the sample is examined for the presence of the expected probe ligation product.

Array Techniques Techniques that utilize hybridization to surface-bound DNA probe arrays are useful for analyzing the nucleotide sequence of target nucleic acids. Such techniques involve two bonds via hydrogen bonding according to Watson-Crick base pairing rules.
It relies on the inherent ability of nucleic acids to form main strands. In theory, and to some extent practical, hybridization to a surface-bound DNA probe array can provide a relatively large amount of information in a single experiment. For example, array techniques have identified single nucleotide polymorphisms in relatively long (1,000 residues or bases) sequences (Kozal, M., et al., Nature Med. 7: 753-759, Ju
ly 1996). In addition, array technology is also useful for analyzing certain gene expressions by comparative analysis of complex mixtures of mRNA target sequences (Lockart, D., et al.
l., (1995) Nat. Biotech 14: 1675-1680). Array technology offers reasonable sensitivity and accuracy when performed on specific applications or specific sets of target sequences, but allows multiple and / or different targets to be applied simultaneously. It cannot be comprehensive. This is largely due to the relatively long probe sequences required to form the probe / target duplex and then be detected.
In addition, the use of this relatively long probe results in one nucleotide difference due to the inherently small thermodynamic difference between the perfectly complementary and one mismatch in the probe / target duplex. It will be difficult to find. Further, detection relies on solution diffusion properties and hydrogen bonding between complementary target and probe sequences.

Mass Spectrometry Technology Mass Spectrometry (MS) is a powerful tool for analyzing complex mixtures of compounds, including nucleic acids. In addition to accurately determining the raw mass, several alternative MS strategies can provide primary structure information. The use of MS for DNA analysis has potential applications in detecting DNA modifications, determining the mass of DNA fragments, and sequencing DNA (see, eg, Fields, GB, Clinical Chemistry 43: 1108 (1997)). have. Fast atom bomb mass spectrometry (Fast atom bombar
dment; FAB) and electrospray ionization (eclectrospray ionizatio)
n; ESI) Both collision-induced dissociation / tandem MS have been applied to identify DNA modification sites.

Although MS is a powerful tool for analyzing complex mixtures of related compounds, including nucleic acids, utilizing it to analyze the sequence of nucleic acids depends on the available ionization and detection methods. It is limited. For example, ESI
The analyzer creates a distribution of highly charged ions with a mass to charge ratio within the range of a commercially available quadrupole mass spectrometer. ESI is sensitive and requires only femtomolar amounts of sample, but relies on complex charges to achieve sufficient ionization,
Even for simple nucleic acids, it produces complex, complex-to-interpretation charged spectra.

[0012] Matrix-assisted laser desorption ionization used in connection with a time of flight (TOF) mass spectrometer
ion; MALDI) has a relatively wide mass range, high resolution (m / Δm ≦ 1.0 at 5,000 mass) and sampling rate (up to 1 sample per second), so that the sequence of nucleic acid The decision has great potential. In one aspect, MALDI offers the advantage over ESI and FAB of ionizing and easily analyzing large mass biomolecules. In addition, compared to ESI, MALDI is a largely single-charged species.

However, in general, DNA analysis by MALDI may suffer from insufficient resolution of high molecular weight DNA fragments, instability of DNA, and interference by sample preparation reagents. Because MALDI gives large kinetic energies to high molecular weight ions, the longer the oligonucleotide, the broader and less intense the signal. Although potentially used to analyze high molecular weight nucleic acids, MALDI-TOF cuts the nucleic acid backbone, which further complicates the spectrum. As a result, MALDI-TOF
The length of the nucleic acid sequence that can be analyzed at present is limited to about 100 bases or 100 residues. Wang et al. (WO 98/03684) makes good use of "material fragmentation" and combines it with delayed pulsed ion extraction to determine the sequence of nucleic acid analytes.

[0014] Numerous methods have been disclosed for the successful use of standard sequencing methods to generate target fragments for analysis by mass spectrometry. For example, US Pat. No. 5,288,
No. 644 (Beavis et al.), US Pat. No. 5,547,835 (Koster), and US Pat. No. 5,622,824 (Koster) disclose exonuclease digestion or S
Ladder-like target M generated by either of anger's standard sequencing methods
A method for determining the sequence of a target nucleic acid using ALDI-TOF is disclosed. Beavis
Discuss DNA sequencing methods that utilize different base-specific reactions using different sets of DNA fragments to produce pieces of DNA with unknown sequence. DN
Each of the different sets of A fragments has a common identity and is terminated at a particular base by an unknown sequence. The molecular weights of the different sets of DNA fragments are each measured with a MALDI mass spectrometer and then used to deduce the DNA sequence.

[0015] Koster used Sanger's sequencing strategy to analyze nested fragments obtained by base-specific chain termination through various molecular weights using mass spectrometry such as MALDI or ESI mass spectrometry. By doing so, the sequence information is assembled. This method has been linked to solid phase sequencing in which the template is labeled with biotin and bound to streptavidin-coated magnetic beads. By this method, the sequence of exons 5 and 8 of the p53 gene could be determined using 21 primers (Fu et al., Nat, Biotechnol 16: 381 (1998). Oligonucleotide primers, chain terminating nucleotides) Processing by introducing mass alterations in triphosphates and / or chain-extending nucleotide triphosphates, and by using an integrated tag sequence that can be diversified by hybridizing the differentiated mass with a tag-specific probe Ability (US Patent No. 5
, 547,835). However, it is important to note that all such methods require some prior knowledge of the target sequence or the introduction of a known sequence to act as a primer binding site.

[0016] Enzyme assays that determine the presence, location and identity of a mutation in a sequence that is known separately, such that at least some information is known a priori as to the presence, location and / or identity of the mutation Attempts have been made to use mass spectrometry together with. For example, US Pat. No. 5,605,798 discloses that a DNA primer complementary to a known target molecule adjacent to a known region of interest is extended by a DNA polymerase in the presence of a mass-tagged dideoxynucleotide. Methods are disclosed. The identity of the mutation is then determined by analyzing the mass of the dideoxy-extended DNA. The diversification method may be useful for detecting potential mutations / mutations at defined sites by extension by dideoxynucleotides, and at the same time determining which specific dideoxynucleotides have been incorporated. It has been revealed.

Attempts have been made to address the shortcomings described above in the mass spectrometric analysis of nucleic acids. For example, Gut (WO 96/28691) discloses a method for altering the exchange properties of phosphodiester phosphodiesters of nucleic acids to make them more suitable for MS analysis. Methods for introducing modified nucleotides that stabilize nucleic acids against fragmentation have also been described (Schneider and Chait, Nucleic Acids Res, 23, 1570 (1995),
Tang et al., J Am. Soc. Mass Spectrom. 8, 218-224, 1997).

To address some of the deficiencies mentioned above, non-cleavable mass tags have also been utilized. For example, Japanese Patent No. 59-131909 discloses the design of a mass spectrometry method to detect nucleic acid fragments fractionated by electrophoresis, liquid chromatography and high-speed gel filtration, where atoms are incorporated into nucleic acids. . Usually, the atoms that are not present in nucleic acids are sulfur, bromine, iodine, silver, gold, platinum and mercury.

To circumvent some of the problems associated with MS analysis of nucleic acids, cleavable mass tags have been utilized. For example, PCT application WO 95/04160 (Southern, e
t al.) employs a target-mediated ligation between a cleavable mass-tagged oligonucleotide containing a reporter group using mass spectroscopy techniques and a DNA-bound protein attached surface. An indirect method for analyzing the sequence of a target nucleic acid is disclosed.
The sequence to be determined is first hybridized to an oligonucleotide attached to a solid support. The solid support with the hybrid described above is incubated with a solution of the encoded oligonucleotide reagent forming a library containing any sequence of any length. A ligase is provided to allow the oligonucleotide on the support to ligate to members of the library that hybridize with the target adjacent to the oligonucleotide. Unbound reagents are removed by washing.
The linker, which is part of the library member linked to the oligonucleotide, breaks and removes the tag. It is collected and analyzed by mass spectrometry.

The focus common to the above described techniques is that the target site (either between or within targets) can be determined in a single determination where some portion of the target sequence is known.
Is to provide a way to increase the number of This theme of multiplexing is directly stated or implied in the teachings of the above-mentioned patent application. The use of one or more oligonucleotides as hybridization probes or primers for extension or ligation is defined by the sequence surrounding the site of interest, and is therefore a specific application. Thus, Southern
The oligonucleotide reagents described above, with the exception of the mass tag technology disclosed by R.G., are not general in terms of target sequence and must be made for each defined application. As such, the number of different oligonucleotides used in multiplexing studies is generally only a small subset of the theoretically complete set. The ratio of the actual sequence coverage provided by a particular oligonucleotide mixture to the theoretical coverage provided by the complete set is the mixture coverage complexity (the mix
ture coverage complexity) (see discussion below). For example,
In many of the methods described (ie, US Pat. No. 5,605,798,
(WO 92/15712 and WO 97/35033), the length of the probe varies from 8 to 20 nucleotides depending on the specific application and detection method.
The number of probes in the complete sequence set can be described by equation 4 ^L , where L
Is equal to the length of the probe. Therefore octamer nucleotides probe sequence will have a member of 4 ⁸ or 65,536 a complete set. If the number of sites to be examined in the multiplex determination is about 500, which is a reasonable upper limit on the number of oligonucleotide probes in a single determination for a technique of the type described above, the nucleotide of the octamer being examined The mixture coverage complexity of the probe mixture (see discussion below) is 500 /
65,536 or about 1/130. However, in most cases, probes are 15-20 nucleotides in length. This increase in length guarantees the specificity of the probe for a particular target sequence, but significantly reduces the mixture coverage complexity of the probe mixture. Thus, for the multiplexed methods and types of applications described above, the oligonucleotide mixture under investigation is not designed to complete the sequence with respect to target sequence coverage and therefore cannot be considered a common reagent. Was.

The purpose of many array-based sequencing methods is to determine the content of “short words” in the target nucleic acid sequence, ie, all oligonucleotide sequences present. For example, in a technique using hybridization to a DNA probe array bound to a surface, a set of oligonucleotides of a specific length are arranged at spatially different positions on a substrate to form an array, and a target sequence is defined as an array. Hybrids can be formed. (See, for example, US Pat. Nos. 5,202,231, 5,492,806, and 5,695,940)
. The target sequence binds to a site containing a short word that is complementary to one of the short words in the sequence. Other methods for probing surface-bound targets with a continuous set of oligonucleotide probes have been disclosed (eg, US Pat. No. 5,20).
No. 2,231, U.S. Pat. No. 5,492,806, and U.S. Pat. No. 5,695.
940). By identifying the location of hybridization via fluorescence measurements or the like, or knowing its identity with the oligonucleotide probe, the exact short word content of the target nucleic acid sequence can be theoretically determined. This information can then be used to reconstruct the sequence of the target nucleic acid (eg,
Pevzner, PA, J. Biomolecular Structure Dynamics 7, 63 (1989), Pevzner,
PA, et al., J. Biomolecular Structure Dynamics 9, 399 (1991), Ukkonen,
E., Theoretical Computer Science 92, 191 (1992)). However,
It is important to emphasize that in order to generally determine the content of short words in unknown targets, a relatively complete set of oligonucleotide probe sequences is required.

Techniques such as identifying short word content in a target nucleic acid sequence are useful for applications such as novel sequencing, resequencing, detecting mutations, and detecting changes due to mutations. As the length of the target sequence increases, the success rate, or success rate with which it can be analyzed, decreases. Certain applications, such as mutation detection, require only qualitative information, so their success rate is lower than that of applications that require quantitative information, such as novel sequencing. Generally high. For example, the presence of a few word repeats will greatly reduce the success rate of new sequencing, but will not significantly affect the success rate for detecting mutations. In other applications,
Substantial prior information is available to help interpret the content of the short word, thus increasing the success rate of the result.

It is an object of the present invention to determine the content of short words in a target nucleic acid using mass spectrometry. However, the success rate of such analyzes is expected to be relatively low, as the presence of a particular mass in the mass spectrum only indicates that one of many potential nucleic acids is present. ing. For example, using only natural nucleotides, the sequence GGCTTTA is distinguished from the sequence GCTTTAG by mass, and the presence of a mass peak at 2,142 atomic mass units indicates that at least three T and two G in the mixture. It simply indicates that there is one nucleic acid sequence consisting of one A and one C. Ambiguity is further confused by the coincidence of masses. For example, the mass peak at 2,193 is
It may contain information from a nucleic acid sequence containing 6 A and 1 T, or 1 A, 2 C, 3 G and 1 T. It is an object of the present invention to reduce such type ambiguity in the content of short words in target nucleic acid sequences.

SUMMARY OF THE INVENTION The present invention relates to reagents and methods for reproducing target nucleic acids in short word shapes that can be analyzed by high resolution mass spectrometry techniques. Reagents and methods utilize common oligonucleotide precursor mixtures (precursor mixtures of X-mers) and enzymatic processes to make such X-mer nucleotides complementary to the target nucleic acid in a defined mixture. Only the length of the precursor is changed, and the mass is concomitantly changed.

One aspect of the present invention is a mixture for directly analyzing a mass spectrum of a nucleic acid. The mixture includes natural and precursors of X-mer nucleotides with altered mass, having a minimum of 3 nucleotides. Minimum mixture coverage complexity (C
_CM ) is obtained by dividing 56 by the number of different X-mer nucleotides in the mixture. The length of the X-mer nucleotide precursor can be independently selected for each X-mer nucleotide precursor. The mass number complexity (MNC) of the mixture is greater than the mass number complexity of any natural equivalent of the mixture. X in the mixture
Each of the dimeric nucleotide precursors is represented by one chemical species.

Another aspect of the invention is a method for analyzing a target nucleic acid sequence. The mixture of X-mer nucleotide precursors is hybridized to the target nucleic acid sequence. The mixture is
Includes natural and mass-modified X-mer nucleotide precursors with a minimum of 3 nucleotides and has the minimum mixture coverage complexity obtained by dividing 56 by the number of different X-mers in the mixture . The length of the X-mer nucleotide precursor can be independently selected for each X-mer nucleotide precursor. The X-mer nucleotide precursors in the mixture are each represented by one chemical species. The hybrid is processed to alter the mass of the X-mer nucleotide precursor of the hybrid in a target sequence mediated reaction. Analyze the previous product by mass spectrometry.

Another aspect of the present invention relates to a method for analyzing a target nucleic acid sequence having a 3 ′ end and a 5 ′ end. The target nucleic acid sequence hybridizes with the various nucleic acid probes on the array. The array includes a surface and various nucleic acid sequence probes. The probes each had a cleavable linker attached to the surface and 3 ′ and 5 ′ ends.
It comprises a nucleic acid sequence having a phosphate, the 3 'end of the nucleic acid being attached to a cleavable linker. The mixture of X-mer nucleotide precursors is hybridized to the target nucleic acid sequence. The mixture has native and mass-modified X-mer nucleotide precursors with a minimum of 3 nucleotides and the minimum mixture coverage obtained by dividing 56 by the number of different X-mer nucleotides in the mixture. Has complexity. The length of the X-mer nucleotide precursor can be independently selected for each X-mer nucleotide precursor. The X-mer nucleotide precursor in the mixture is 1
Represented by two chemical species. The X-mer nucleotide precursor hybridized at a position adjacent to the terminal 5 'phosphate is linked to a surface-bound probe to form a hybridized precursor / probe complex with the target nucleic acid sequence attached thereto. . The complex is cleaved with a cleavable linker and analyzed by mass spectrometry.

Another aspect of the invention is a kit for performing the above method. The kit comprises various nucleotides selected from the group consisting of a mixture as described above, an enzyme having DNA polymerase activity, and a natural chain-terminated triphosphate.

Another embodiment of the present invention is a kit for performing the above method. The kit comprises various nucleotides selected from the group consisting of the mixture described above, an enzyme having DNA polymerase activity, and a chain-terminated triphosphate with altered mass.

[0030] Another embodiment of the present invention is a kit for performing the above method. The kit comprises the mixture described above, an enzyme having DNA polymerase activity, various nucleotides selected from the group consisting of natural chain-terminated triphosphates, and various extended nucleotide triphosphates.

Another embodiment of the present invention is a kit for performing the above method. The kit comprises the mixture described above, an enzyme having DNA polymerase activity, various nucleotides selected from the group consisting of altered mass, chain terminated triphosphates, and various extended nucleotide triphosphates.

[0032] Another embodiment of the invention is a kit for performing the above method. The kit comprises a mixture as described above, an enzyme having DNA polymerase activity, a modified nucleotide, a variety of nucleotides selected from the group consisting of chain-terminated triphosphates, a variety of extended nucleotide triphosphates, and a nuclease. Become.

[0033] Another embodiment of the invention is a kit for performing the above method. The kit comprises a mixture as described above, an enzyme having DNA polymerase activity, various nucleotides selected from the group consisting of natural and thiophosphate-extended nucleotide triphosphates, and a chain-terminated triphosphate with altered mass. It becomes.

[0034] Another embodiment of the invention is a kit for performing the above method. The kit comprises a mixture as described above, an enzyme having DNA polymerase activity, various nucleotides selected from the group consisting of native and thiophosphate-extended nucleotide triphosphates, a chain-terminated triphosphate with altered mass, and 5 'Exonuclease.

[0035] Another embodiment of the invention is a kit for performing the above method. The kit comprises the mixture as described above and DNA ligase.

[0036] Another embodiment of the invention is a kit for performing the above method. The kit comprises a mixture as described above and a condensing agent.

[0037] Another embodiment of the invention is a kit for performing the above method. The kit comprises a mixture as described above, a DNA ligase, and an array, wherein the array comprises a surface, a cleavable linker attached to the surface, and various nucleic acid sequences having a 3 'end and a terminal 5' phosphate. And a probe, wherein the 3 'end of the nucleic acid sequence is attached to a cleavable linker.

[0038] Another embodiment of the invention is a kit for performing the above method. The kit comprises a mixture, a concentrating agent, and an array as described above, the array having a surface, a cleavable linker attached to the surface, and a nucleic acid sequence having a 3 'end and a terminal 5' phosphate. It contains a nucleic acid sequence probe and the 3 'end of the nucleic acid sequence is attached to a cleavable linker.

DETAILED DESCRIPTION OF THE INVENTION Definitions In this specification and the claims that follow, reference is made to a number of terms that are to be defined to have the following meanings.

The term “polynucleotide” or “nucleic acid” refers to a compound or composition that is a nucleotide or nucleic acid polymer. The polynucleotide may be a natural or synthetic compound. Polynucleotides can have from about 20 to 5,000,000 or more nucleotides. Larger polynucleotides are generally found in nature. As isolated, the polynucleotide has about 30 to 50,000 nucleotides, and typically about 100 to 20,000 nucleotides.
It can have 000 nucleotides, and more often from 500 to 10,000 nucleotides. Thus, it is clear that fragmentation often occurs in the isolation of polynucleotides from the natural state. Fragmentation of long target nucleic acid sequences, particularly RNA, prior to hybridization is useful for reducing competitive intramolecular structures.

Polynucleotides include tRNA, mRNA, rRNA, mitochondrial DNA
RNA, genes, chromosomes, plasmids, cosmids, and microorganisms, including chloroplast DNA and RNA, DNA / RNA hybrids, or mixtures thereof;
For example, purified or unpurified, including DNA (dsDNA and ssDNA) and RNA containing the genome of biological materials such as bacteria, yeast, phage, chromosomes, viruses, viroids, molds, fungi, plants, animal humans, etc. Includes nucleic acids and fragments thereof from any source material. Polynucleotides are only a small fraction of a complex mixture, such as a biological sample. Also included are genes such as the hemoglobin gene for sickle cell anemia, cystic fibrosis gene, oncogene, cDNA and the like.

[0042] Polynucleotides can be obtained from various biological sources by methods well known in the art. A polynucleotide is cleaved at an appropriate site, for example, by treatment with a restriction endonuclease or a site-specific chemical cleavage method,
A fragment containing the target nucleotide sequence may be obtained.

For the purposes of the invention, polynucleotides or truncated fragments obtained from polynucleotides are typically at least partially denatured, single stranded, or treated to be denatured or single stranded. do. Such treatments are well known in the art and include, for example, heat or alkali treatment, or single-stranded enzymatic degradation. For example, a denatured material can be made by heating dsDNA at 90 ° C. to 100 ° C. for about 1 to 10 minutes.

[0044] Nucleic acids can be used in polymerase chain reaction (PCR), asymmetric PCR, ligase chain reaction (L
CR) and the like, and may be made by in vitro replication and / or amplification methods. The nucleic acid may be single-stranded or double-stranded. Single-stranded nucleic acids are preferred in the hybridization step of the method of the invention because they lack complementary strands that compete for the oligonucleotide precursor.

The phrase “target nucleic acid sequence” generally refers to the sequence of a nucleic acid to be identified, detected, or analyzed, contained in part or all of a polynucleotide. In the present invention, the identity of the target nucleotide sequence may or may not be known. In order to be able to sufficiently prepare various sequences capable of hybridizing with the target nucleotide sequence, and various sequences of oligonucleotides such as probes and primers, and other molecules necessary for performing the method according to the present invention, etc. The identity of the target nucleotide sequence may be known to some extent.

The target sequence typically contains from about 30 to 5,000 or more nucleotides, and preferably contains from 50 to 1,000 nucleotides. The target nucleotide sequence is generally a fraction of a large molecule, or may be a molecule of the entire polynucleotide, substantially as described above. The minimum number of nucleotides in the target nucleotide sequence is selected such that the presence of the target polynucleotide in the sample is a specific indicator of the presence of the polynucleotide in the sample. The maximum number of nucleotides in a target nucleotide sequence usually depends on several factors. That is, the length of the polynucleotide from which it is derived, the fragility of such polynucleotides during cleavage or other steps during isolation, the processing efficiencies required for sample preparation for analysis (eg, from DNA template to RNA Transcription, and efficiency in identifying, detecting, amplifying, and / or otherwise analyzing the target nucleotide sequence where appropriate.

The term “oligonucleotide” refers to a polynucleotide, which is typically a single-stranded, synthetic polynucleotide, but may be a naturally occurring polynucleotide. The length of the oligonucleotide will generally be, for example, probe, primer,
It depends on its special role, such as X-mer nucleotides. Various techniques can be used to prepare the oligonucleotide. Such oligonucleotides can be obtained by biological or chemical synthesis. For short oligonucleotides (up to about 100 nucleotides), chemical synthesis is often more economical than biological synthesis. In addition to economics, chemical synthesis provides a convenient way to incorporate low molecular weight compounds and / or modified bases in particular synthetic steps. Furthermore, chemical synthesis is extremely flexible in selecting the length and region of the target polynucleotide binding sequence. Oligonucleotides can be synthesized by standard methods, such as those used in commercially available automated nucleic acid synthesizers. When DNA is chemically synthesized on appropriately modified glass or resin, DNA covalently bonded to the surface can be obtained. This provides advantages for washing and boiling the sample. Oligonucleotide synthesis methods include phosphoramidate technology (Caruthers, MH, et al., "Methods in Enzymology" Vol. 154, pp287).
-314 (1988)) and the phosphate triester and phosphate diester methods (Narang, et al.).
al., (1979) Meth. Enzymol. 68:90), and synthesis on a support (Beaucage, et a
l., (1981) Tetrahedron Letters 22: 1859-1862), and "Synthesis and Appl.
ication of DAN and RNA "SA Narang, editor, Academic Press, New York,
These references, among others, described in 1987, are hereby incorporated by reference. The method of chemically synthesizing a spatially tractable array of oligonucleotides bound to a glass surface via photolithography is described in AC Pease et.
Natl. Acad. Sci. USA (1994) 91: 5022-5026.

The term “X-mer nucleotide” refers to a defined length, typically at least 3
Refers to an oligonucleotide having a length of 4 nucleotides, preferably 4 to 14 nucleotides and usually 5 to 7 nucleotides.

The phrase “X-mer nucleotide precursor” refers to “oligonucleotide precursor” with reference to a nucleic acid sequence that is complementary to a portion of the target nucleic acid sequence. The oligonucleotide precursor may be a sequence of nucleoside monomers linked by a phosphorus bridge (eg, phosphodiester, alkyl and allyl phosphates, phosphorothionates, phosphate triesters) or non-phosphorus bridges (eg, peptides, sulfamic acid, etc.). is there. They include circular, linear, or linear single-stranded DNAs and single-stranded DNAs.
The natural or synthetic molecule of the present stranded RNA may optionally include domains that stabilize the secondary structure (eg, stem and loop, and loop-stem-loop structures). The oligonucleotide precursor contains a 3 'end and a 5' end. This phrase is represented by ω.

The term “mixture” refers to a physical mixture of two or more substances. This term is represented by Ω.

The phrase “oligonucleotide probe” refers to an oligonucleotide used to bind another oligonucleotide or a portion of a polynucleotide, such as a target nucleotide sequence. The design and construction of oligonucleotide probes generally depends on the sequence to which they bind.

The phrase “oligonucleotide primer” generally refers to an oligonucleotide used for chain extension of a polynucleotide template, for example, in the amplification of nucleic acids. Oligonucleotide primers contain a sequence at the 3 'end that allows hybridization to a defined sequence of the target polynucleotide, typically 1
It is a single-stranded synthetic nucleotide. Typically, oligonucleotide primers have at least 80%, preferably 90%, more preferably 95%, and most preferably 100% complementarity to a defined sequence or primer binding site. With respect to the number of nucleotides in the sequence to which the oligonucleotide primer can hybridize, the stringency conditions under which the oligonucleotide primer will hybridize should prevent excessive random non-specific hybridization. Typically, the number of nucleotides in the oligonucleotide primer is at least as many as the defined nucleotides of the target polynucleotide, ie, at least 10 nucleotides, preferably 15 nucleotides, generally 10 to 200, Preferably it is 20 to 50 nucleotides.

The phrase “nucleoside triphosphate” refers to a nucleoside having a 5′-triphosphate substituent. Nucleosides are pentose derivatives in which the base containing the nitrogen of the purine or pyrimidine derivative is covalently bonded to the 1 'carbon atom of the pentose, typically deoxyribose or ribose. Purine bases include adenine (A
), Guanine (G), inosine (I) and its derivatives and analogs. Pyrimidine bases include cytosine (C), thymine (T), uracil (U) and derivatives and analogs thereof. Nucleoside triphosphates include four common deoxyribonucleoside triphosphates, deoxyribonucleoside triphosphates such as dATP, dCTP, dGTP and dTTP, and four common triphosphates, r
Ribonucleoside triphosphates such as ATP, rCTP, rGTP and rTTP are included. The term "nucleoside triphosphate" also includes derivatives and analogs thereof, examples of which include derivatives that are recognized and polymerized in a manner similar to underivatized nucleoside triphosphates. .

The term “nucleotide” or “nucleotide base” or “base” refers to a nucleic acid polymer, a base-sugar-phosphate combination that is a monomer of DNA and RNA. The terms used herein include modified nucleotides as defined below. In general, the term refers to a cyclic furanose glycoside form that is phosphorylated at the 5 ′ position and has either a purine or pyrimidine base at the C-1 ′ position of the sugar via a β-glycosyl C1′-N bond. Sugar (RN
(A-D-ribose in A and β-D-2'deoxyribose in DNA). Such terms are interchangeable and are denoted by b. Nucleotides include, inter alia, modified bases (eg, methylcytosine) and modified sugars (eg, 2′-O-methylribosyl, 2′-O-methylethylribosyl, 2′-fluororibosyl, 2′-aminoribosyl) And the like, and may include a nucleotide having a modified mass, including a nucleotide having a nucleoside modified with a nucleoside, and may be a natural or synthetic nucleotide.

[0055] The term "DNA" refers to deoxyribonucleic acid.

The term “RNA” refers to ribonucleic acid.

The term “natural nucleotide” is defined as a cell that contains deoxycytidylic acid (pdC), deoxyadenylic acid (pdA), deoxyguanylic acid (pdG), and deoxythymidylic acid (pdT). To form the basic structure of sex DNA, and deoxycytidylic acid (pdC), deoxyadenylic acid (pd
A) Refers to nucleotides that form the basic structure of cellular RNA defined to include deoxyguanylic acid (pdG) and deoxyuridylic acid (pdU). pdU is considered to be the natural equivalent of pdT.

The term “natural nucleotide bases” refers to purine and pyrimidine-type bases found in cellular DNA and RNA, including cytosine (C), adenine (A), guanine ( G) and thymine (T), and cellular RNAs include (C), adenine (A), guanine (G) and uracil (U). U is considered to be the natural equivalent of T.

The phrase “modified nucleotide” refers to a unit in a nucleic acid polymer that includes a modified base, sugar, or phosphate. Modified nucleotides can be made as part of the nucleic acid polymer or by chemically modifying the nucleotide prior to incorporating the modified nucleotide into the nucleic acid polymer. For example, the methods described above for oligonucleotide synthesis may be used. Alternatively, modified nucleotides can be made by incorporating the modified nucleoside triphosphate into the polymer strand during the amplification reaction. Examples of modified nucleotides are for illustrative purposes and are not intended to be limiting, but dideoxynucleotides, biotinylated derivatives and analogs,
Includes amine modifications, alkylations, fluorescent labels, etc., and also includes phosphorothioates, phosphites, derivatives modified with ring atoms, and the like.

The term “Watson-Crick base pair” refers to “Principles of Nucleic Acid
Structure "; Wolfran Saenger, Springer-Verlag, Berlin (1984) that the two bases are hydrogen-bonded by a special pattern of hydrogen-bonded donor and acceptor hydrogen bonding that has the canonical rule defined by Berlin. Say.

The phrase “base pair specificity” of nucleotide base b refers to a number of naturally occurring nucleotide bases such that the bases form Watson-Crick base pairs. This term is denoted by S _bp (b). For example, S _bp (b) for four natural nucleotides is as follows: S _bp (A) = 1, S _bp (G) = 1, S _bp (C) = 1, and S _bp ( T) = 1.

The phrase “natural complement of nucleotides” refers to natural bases that allow nucleotides to form base pairs most preferably according to Watson-Crick base pairing rules. A nucleotide has multiple natural base pair complements if it can base pair with the same affinity as multiple natural bases, or can form the best base pair with another natural base pair in another environment. Conceivable.

The phrase “natural equivalent of a nucleotide” refers to the natural complement of a natural complement of a nucleotide. If the nucleotide has more than one natural complement,
It is believed to have multiple natural equivalents.

The phrase “natural equivalent of an oligonucleotide precursor” refers to an oligonucleotide precursor in which each nucleotide has been replaced with a natural nucleotide equivalent. If one or more of the original nucleotides has more than one natural equivalent, the oligonucleotide precursor is considered to have more than one natural equivalent, and the equivalent is selected from any combination that can be substituted. Words are denoted by NE (ω).

The term “nucleoside” is a nucleotide that lacks a base-sugar combination or a phosphate moiety.

“Chain-terminated nucleoside triphosphate” refers to a nucleoside triphosphate that can be added to an oligonucleotide primer in a chain extension reaction, but cannot undergo chain extension. By way of example, and not by way of limitation, four standard dideoxynucleotide triphosphates, analogs of modified dideoxynucleotide triphosphates, thio analogs of natural and modified dideoxynucleotide triphosphates , Arabinose, 3'-amino, 3'-azide, 3'-fluoro derivatives and the like.

The phrase “dideoxynucleotide triphosphate” refers to the four basic natural dideoxynucleotide triphosphates (ddATP, ddGTP, dd for DNA)
DdATP, ddGTP, d for CTP and ddTTP, and RNA
dCTP and ddUTP) and dideoxynucleotide triphosphates with altered masses and include these. This term is represented by ddNTP.

The phrase “extended nucleoside triphosphate” refers to natural deoxynucleoside triphosphates, modified deoxynucleotide triphosphates, modified mass deoxynucleoside triphosphates, 5 ′ (α) phosphothioate, and related U.S. Pat. No. 5,171,534 and U.S. Pat. No. 5,171,534, the portions of which are incorporated herein by reference.
And 5'-N (α-phosphoramidate) analogs, such as natural and altered mass deoxy and ribonucleoside triphosphates, as disclosed in US Pat. I have.

The phrase “nucleotide polymerase” refers to a DN whose extension is complementary to them.
A or A: A catalyst for forming a polynucleotide extension along an RNA template, usually an enzyme. Nucleotide polymerases are template-dependent polynucleotide polymerases that utilize nucleoside triphosphates as the basic backbone and extend the 3 'end of the polynucleotide to provide a sequence complementary to the polynucleotide template. Typically, the catalyst is an enzyme such as a DNA polymerase, for example, prokaryotic DNA polymerase (I, II, or III), T4 DNA polymerase, T7 DNA polymerase, E. coli DNA polymerase (Klenow fragment 3'-5'exo), reverse transcription. Enzyme, Vent DNA polymerase, Pfu DNA
Polymerase, Taq DNA polymerase, Bst DNA polymerase, etc.
Alternatively, it is an RNA polymerase such as T3 and T7 RNA polymerase. The polymerase enzyme may be obtained from any source, such as cells, bacteria such as E. coli, plants, animals, viruses, thermophilic bacteria, and the like.

“Amplification” of nucleic acids and polynucleotides refers to the formation of one or more copies of a nucleic acid or polynucleotide molecule (exponential amplification), or one or more copies of the complement of a nucleic acid or polynucleotide alone (linear). Amplification). Methods of amplification include the polymerase chain reaction (PCR), which is based on the repetition of a cycle of denaturation, annealing of oligonucleotide primers, and extension of primers, resulting in the flanking of primers by a thermophilic template-dependent polynucleotide polymerase. To obtain an exponential increase in the copy of the desired sequence in the isolated polynucleotide sample. Annealing facing each other with DNA strands 2
The two different primers were placed and separated by a distance defined by the distance between the 5 'ends of the oligonucleotide primers, such that one primer product catalyzed and extended by the polymerase served as the template strand for the other. Accumulate the main chain fragment. Reagents for performing such amplification include oligonucleotide primers, nucleotide polymerases such as deoxyadenosine triphosphate (dATP),
Deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCT
P) and nucleoside triphosphates such as deoxythymidine triphosphate (dTTP). Other amplification methods include single-stranded polynucleotide amplification using one oligonucleotide primer, ligase chain reaction (LCR), amplification-based nucleic acid sequencing (NASBA), Q-beta-replicase and 3SR. included.

In the context of nucleotide sequences, the terms “hybridize (hybridize)” and “bind” are used interchangeably herein. The ability of two nucleotide sequences to hybridize to each other is based on the degree of complementarity of the two nucleotide sequences, in other words,
Based on fractionation of matched complementary nucleotide pairs. The more complementary a nucleotide in any sequence is to another sequence, the more it will be able to hybridize under stringent conditions and the more specific the binding of the two sequences will be. Stringency can be increased by increasing the temperature, increasing the proportion of co-solvent, decreasing the salt concentration, and the like.

The terms “complementary,” “complement,” or “complementary nucleic acid sequence” refer to a nucleic acid strand that is related to a base sequence in another nucleic acid strand by Watson-Crick base pairing rules. . In general, two sequences are complementary if one sequence can be joined in the antiparallel sense to the other sequence, wherein the 3 'end is each linked to the 5' end of the other sequence and A, T (U), G, and C of the sequence are arranged as T (U), A, C, and G, respectively, in the other sequence. The RNA sequence can also include complementary G / U or U / G base pairs.

The term “hybrid” refers to a double-stranded nucleic acid molecule formed by hydrogen bonding between complementary nucleotides. The term "hybridizes" refers to the process by which single-stranded nucleic acid sequences form double helical segments via hydrogen bonding between complementary nucleotides.

The term “altered mass” refers to an internal change to the chemical structure, ie, due to the addition, deletion, or substitution of a chemical moiety, or an external change to the chemical structure, ie, a covalent bond. Chemical moiety (atom or molecule)
Refers to a nucleic acid sequence whose mass has been changed by either of the above.

The phrase “atomic mass number” refers to the nucleon number of the most common isotope of the element of interest.

The reported mass for all nucleic acids (ie, nucleotides, nucleotide precursors, oligonucleotides, X-mer nucleotides and X-mer nucleotide products) is the highest abundant isotope of the constituent atoms (ie, C12, N14, O16
, P31, I127) and the protonation state stable in aqueous solution at pH 7 is calculated.

The phrase “mass number of oligonucleotide precursor” refers to the sum of the mass numbers of atoms constituting the oligonucleotide precursor. This phrase is represented by z (ω).

The phrase “mass number histogram of the oligonucleotide precursor mixture” Ω
A function h from natural number to natural number defined by h (z).
h (z) is the number of oligonucleotide precursors in the mixture Ω such that z (ω) = z.

The phrase “average ambiguity of the oligonucleotide mixture” (A (Ω)) is the sum of the squares of the mass number histogram values of the mixture of oligonucleotide precursors, given by the number of oligonucleotide precursors in the mixture. It is divided and may be expressed mathematically as follows.

[0080]

(Equation 1)

The phrase “mass number complexity” (MNC) refers to the number of oligonucleotide precursors in a mixture divided by the average ambiguity of the mixture of oligonucleotide precursors, and is mathematically described as May be expressed as

[0082]

(Equation 2)

“Oligonucleotide coverage complexi
ty) "CC ₀ (ω) may be mathematically expressed as:

[0084]

(Equation 3)

Here, L is the number of nucleotide bases in the oligonucleotide precursor, and b _i represents the i-th unit of the oligonucleotide precursor.

The phrase “mixture coverage complexity” (CC _M Ω) refers to the sum of the coverage complexity of each oligonucleotide precursor in the mixture, which can be expressed mathematically as Good.

[0087]

(Equation 4)

The term “binning” refers to dividing a mixture into a limited subset, where each individual oligonucleotide in the mixture has at least one oligonucleotide.
In two subset mixtures.

“Composite mixture coverage complexity”
The term "" refers to the coverage complexity in the set of mixtures produced by binning and is equal to the mixture coverage complexity of the original mixture without binning.

The term “complex mass number complexity” refers to the mass number complexity in a set of binned mixtures and is equal to the sum of the mass number complexity in a subset of the mixture.

The phrase “mass spectrum direct analysis” refers to a method of mass spectrometry that analyzes either the target nucleic acid sequence itself or the complement of the target nucleic acid sequence. The target nucleic acid sequence itself or its complement may be altered in mass and include extra nucleotide bases, otherwise the target nucleic acid sequence itself or its complement is actually mass analyzed. On the condition that However, mass spectral analysis, such as analyzing the mass tag portion indicating the presence of the target nucleic acid sequence, such as the indirect method described in PCT application WO 95/04160, is not included in this phrase.

The terms “generality” or “general” when applied to a method refer to a method of mass spectrometry that is applied without reference to specific information. The phrase "positional generality" refers to a method of mass spectrometry that does not require prior information regarding the presence, location or identification of a mutation in a target nucleic acid sequence. The phrase "target generality" refers to mass spectral analysis methods that do not require prior information about the target nucleic acid.

[0093] The term "support" or "surface" refers to a porous or non-porous insoluble material. The surface can have any one of a number of shapes, such as strips, slabs, disks, rods, particles, including beads. The support may be hydrophilic, or may be hydrophilic, inorganic powders such as silica, magnesium sulfate and alumina; natural polymeric materials, especially those made of cellulose, and, for example, filter paper and chromatography paper. Materials derived from cellulose such as paper containing fibers such as nitrocellulose, cellulose acetate, poly (vinyl chloride), polyacrylamide, cross-linked dextran, agarose, polyacrylate, polyethylene, polypropylene, poly (4-methylbutene) Modified synthetic or natural polymers such as polystyrene, polymethacrylate, poly (ethylene terephthalate), nylon, poly (vinyl butyrate); themselves or in combination with others; bioglass, sera Contains glass that can be used as a mix, metal, etc. Natural and synthetic components such as liposomes, phospholipid vesicles and cells can also be used. Binding of the oligonucleotide to the support or surface may be accomplished by techniques well known in the publicly available literature. For example, AC Pease, et al., Proc. Natl. Acad. S
ci. USA, 91: 5022-5026 (1994).

[0094] The term "mutation" refers to a mutation in a nucleotide between two polynucleotides, such as a single nucleotide polymorphism. Generally, mutations occur from individual to individual. A mutation is a change in the nucleotide sequence of a nucleic acid sequence that has been kept normal and will result in the formation of a mutation as differentiated from a normal (unchanged) or wild-type sequence. Mutations can generally be divided into two parts: base pair substitutions and frameshift mutations. The latter results in the insertion or deletion of one to several base pairs. A single nucleotide difference is sufficient to change the phenotype from normal to abnormal, for example, sickle cells.

General Remarks The present invention provides methods and reagents for satisfying the demands for more sensitive, accurate and high throughput in the analysis of target nucleic acid sequences. The methods and reagents may be generally applied to any target nucleic acid sequence and do not require prior information regarding the presence, location or identification of a mutation in the target nucleic acid sequence.

The reagents of the invention are useful for direct analysis of the mass spectrum of nucleic acids and are mixtures comprising natural and altered mass X-mer nucleotide precursors having a minimum length of 3 nucleotides. . The minimum mixture coverage complexity (CC _M ) is 56 divided by the number of different X-mer nucleotides in the mixture. The length of the X-mer nucleotide precursor can be individually selected for each X-mer nucleotide precursor. Each X-mer nucleotide precursor in the mixture is represented by one chemical species.

The methods and reagents of the present invention reduce the ambiguity that exists in mass spectral analysis of target nucleic acid sequences and increase power in any application that uses mass spectrometry to analyze the sequence of a target nucleic acid. This ambiguity reduction is achieved by using a mixture of native and mass-modified oligonucleotide precursors with high levels of mass and coverage complexity. This reduction may be further improved by "binning", ie, using a subset of the reaction mixtures in the at least two reaction mixtures. The results examined separately in a mixture of subsets can also be combined. In this way, in any mass analysis, the degree of overlapping mass between the X-mer nucleotide products is reduced, while maintaining a high level of overall coverage complexity of the target.

The mixtures of the invention are general and universal in the sense that their targets can be used in any of the applications of determining sequence information of a target nucleic acid. In addition, mixtures may be designed for target nucleic acid sequences without reference to prior information, such as the presence, location, or identity of the mutation. However, this does not mean that the mixture is not useful for the analysis of target nucleic acid sequences for which the information is known in advance about the sequences. Moreover, it does not indicate that prior information about the target is not useful for analyzing the resulting mass spectrum.

(Reagent of the Invention) Oligonucleotide (X-mer nucleotide) Precursor If the modified mixture of X-mer nucleotide precursors comprises at least 56 different X-mer nucleotide precursors, the mixture coverage complexity is about 7/8. As the average length of the X-mer nucleotide precursor increases, the number of different X-mer nucleotides in the mixture of the present invention also increases, and the mixture coverage complexity may decrease. The minimum limit of the mixture coverage complexity is equal to 56 divided by the number of X-mer nucleotides in the mixture. The length of the X-mer nucleotide can be individually selected for each X-mer nucleotide.

The specific composition of the mixture is determined on a case-by-case basis and depends on what is required in any given application. The composition of the mixture is defined by the formulas set forth herein. The mixture coverage complexity is defined as:

[0101]

(Equation 5)

Where CC _o is the oligonucleotide coverage complexity of each oligonucleotide precursor, which is defined as:

[0103]

(Equation 6)

Here, L is the number of nucleotide bases in the oligonucleotide precursor, S _bp is the specificity of the base pair, and b _i is the i-th unit of the oligonucleotide precursor.

Examples of mixtures having the above specifications include, but are not limited to, (1) a mixture Ω ₁ containing 56 of the 64 possible trimer nucleotides. (CC _M (Ω ₁ ) = 7/8), (2) a mixture Ω ₂ containing 128 of the 256 possible tetramer nucleotides (CC _M (Ω ₂ ) = １ ／), (3) One possible
The mixture containing 256 species of 024 kinds pentameric nucleotide Ω ₃ (CC _M (Ω ₃
) = 1/4), (4) a mixture Ω ₄ (CC _M (Ω ₄ ) = 1/8) containing 512 of the 4,096 possible hexamer nucleotides, (5) possible 4, A mixture Ω ₅ containing 1,024 of the 096 hexameric nucleotides (CC _M (Ω ₅ ) = 1/4)
(6) a mixture Ω ₆ (CC _M (Ω ₆ ) = 11/64) containing 48 pentameric nucleotides and 512 hexameric nucleotides, (7) 128 pentameric nucleotides and 512 Ω ₇ (CC _M (Ω ₇ ) = 33/128) containing hexameric nucleotides and 128 heptameric nucleotides, (8) 256 pentameric nucleotides and 1,000 hexameric nucleotides There is a mixture Ω ₈ (CC _M (Ω ₈ ) = １ ／) containing nucleotides and 96 heptamer nucleotides.

Examples of mixtures that do not conform to the above specifications include, but are not limited to,
(1) a mixture Ω containing 64 of the 256 possible tetramer nucleotides ₉ (CC_M(Ω₉) = 1/4 <56/64), (2) possible 1,024 pentamers
A mixture Ω containing 128 of the nucleotides_Ten(CC_M(Ω_Ten) = 1/8 <56
/ 128), (3) 384 hexamer nucleotides and 128 heptamer nucleotides
Mixture Ω containing tide₁₁(CC_M(Ω₁₁) = 13/128 <56/512), (4
) 64 pentameric nucleotides, 256 hexameric nucleotides and 64 7-nucleotides
Mixture Ω containing body nucleotides₁₂(CC_M(Ω₁₂) = 33/256 <56/38
4).

Further, the reagents of the invention may comprise natural and altered X-mer nucleotides such that the mass number complexity (MNC) of the mixture is greater than the mass number complexity of any natural equivalent of the mixture. It is a mixture of precursors. The mass number complexity is determined by the X in the mixture.
It refers to the number of multimeric nucleotide precursors divided by the average ambiguity of the X-mer nucleotide precursor mixture, and is mathematically defined as:

[0108]

(Equation 7)

The average ambiguity (A (Ω)) of the X-mer nucleotide precursor mixture is calculated by summing the squared values of the mass number histogram of the X-mer nucleotide precursor mixture and the X-mer nucleotide precursor in the mixture. The value is divided by the number of bodies, and may be expressed mathematically as follows:

[0110]

(Equation 8)

The mass number histogram (h (z)) of the X-mer nucleotide precursor mixture is given by h (
z) is a function h from a natural number to a natural number defined by z). Here, h (z
) Is the number of X-mer nucleotide precursors in the mixture for z (ω) = z.

The MNC of the mixture is typically at least about 2 times, more typically at least about 10 times, and most preferably at least about 50 times greater than the mass number complexity of the natural equivalent to the mixture. For example, the natural hexameric nucleotide, 4,096
The mixture of all species has an MNC of 53 (see discussion below and Table 1). A mixture containing all 4,096 hexameric nucleotides synthesized by permutation combination can have an MNC of 348, which is about 6.5 of its natural equivalent
It is twice. Another mixture in which each X-mer nucleotide was synthesized individually was 55%
It can have an MNC of 9, which is about 10 times its natural equivalent. 4,0
A mixture in which each of the 96 hexameric nucleotides has a unique mass has an MNC of 4,096, about 77 times the natural equivalent.

X-mer nucleotide precursors useful in the methods of the invention have a length of at least 3 nucleotide units. Preferably, the X-mer nucleotide precursor has a length of at least 4 nucleotide units, more preferably at least 5 nucleotide units, most preferably at least 6 nucleotide units. The length of the X-mer nucleotide precursor is individually selected for each X-mer nucleotide precursor in the mixture. Thus, it is possible to have one mixture of X-mer nucleotide precursors with 5, 6, and 7 nucleotides. As can be seen from the above discussion, the values for mixture coverage complexity, and requirements, decrease as the number of X-mer nucleotide precursors increases. If there is more than one length in a mixture, the mixture coverage complexity is obtained by summing the coverage indices of the individual oligonucleotides. So in this case,
The contribution of each oligonucleotide to the mixture coverage complexity depends on its length, with shorter oligonucleotides contributing more. It should be noted that the use of long oligonucleotides can lead to losses in generality. The mixture coverage complexity may be a low value only if a loss in generality is tolerated. Further, the reagents may comprise a mixture of oligonucleotide precursors in sets. In this case, the mixture coverage complexity of any member in the set is lower than above, as long as the overall complexity of the mixture matches above.

[0114] Represented by a number of similar chemical species, such as the ladder-like reporter product used to represent the nucleotide sequence in the oligonucleotides described in PCT application WO 95/04160 (Southern). In contrast, X-mer nucleotide precursors useful in the methods of the invention may each be represented by one species. Thus, while the oligonucleotides in the mixture of WO 95/04160 are associated with a spectrum of masses representing the nucleotide sequence of interest as described above, each of the X-mer nucleotide precursors in the mixture of the invention has one mass. Will have. It is important to recognize that the mass tagging method disclosed by Southern utilizes a cleavable mass tag, in which only the tagged portion of the tagged oligonucleotide is analyzed by mass spectrometry. As seen in the disclosure herein, this is in contrast to the present invention, which relies on a mass spectrum arising from the oligonucleotide product itself, which results from a target-mediated enzymatic process. Further, the mass-modified X-mer nucleotides of the present invention are designed so that any given oligonucleotide sequence has one mass, ie, represents one chemical species. This is different from the mass tag method disclosed by Southern. Due to the design of the "ladder tag" in the Southern method, each distinct oligonucleotide sequence in the mixture is associated with a "spectrum" as a whole mass.

To be useful in the methods of the present invention, it is often desirable to know which X-mer nucleotide precursor is present in the mixture, which is often necessary. However, it is not necessary to know the level of each X-mer nucleotide precursor. As said above, it is advantageous to be able to control the concentration of each X-mer nucleotide in the mixture in order to correct for differences in the thermal stability of the duplex.

In one preferred embodiment, the mixture of precursor X-mer nucleotides is composed of both naturally occurring nucleotides and nucleotides of altered mass. The identity and position of the mass-altered nucleotide in the X-mer nucleotide precursor is
It will depend on a number of factors. These include the desired overall mass complexity in the X-mer nucleotide precursor mixture, the desired thermodynamic properties of the X-mer nucleotide precursor, and the incorporation of altered mass nucleotides into the X-mer nucleotide precursor. Includes the ability of the enzyme or set of enzymes (ie, polymerase and ligase) and the constraints imposed by the particular synthesis of the X-mer nucleotide precursor mixture.

An internal change, ie, the addition, deletion or substitution of a chemical moiety to its chemical structure, or an external change, ie, the addition of a covalently bonded chemical moiety (atom or molecule) to its chemical structure. The mass of the X-mer nucleotide precursor may be changed. One X-mer nucleotide precursor has both internal and chemical moieties, more than one internal change, more than one external change,
Or you may have some of those combinations.

Suitable internal mass alterations include at least one chemical modification to the base pairs of the internucleoside linkages, sugar backbone or X-mer nucleotide precursor. Examples of internally suitable mass-modified X-mer nucleotide precursors include, but are not limited to, 2'-deoxy-5-methylcytidine, 2'-deoxy-5 -Fluorocytidine, 2'-deoxy-5-iodocytidine, 2'-deoxy-5-fluorouridine, 2'-deoxy-5-iodouridine, 2'-O-methyl-5-fluorouridine, 2'-deoxy -5-iodouridine, 2'-deoxy-5 (1-propynyl) uridine, 2'-O-methyl-5 (1-propynyl) uridine, 2-thiothymidine, 4-thiothymidine,
'-Deoxy-5 (1-propynyl) cytidine, 2'-O-methyl-5 (1-propynyl) cytidine, 2'-O-methyladenosine, 2'-deoxy 2,6-diaminopurine, 2'-O -Methyl-2,6-diaminopurine, 2'-deoxy-
7-deazadenosine, 2'-deoxy-6-methyladenosine, 2'-deoxy-8-oxoadenosine, 2'-O-methylguanosine, 2'-deoxy-7-
Deazaguanosine, 2'-deoxy-8-oxoguanosine, 2'-deoxyinosine and the like.

Appropriate external mass changes include mass-tagged X-mer nucleotide precursors or dideoxy terminators. The portion that externally changes the mass may be attached to the 3 ′ end of the X-mer nucleotide, nucleotide base (base), phosphate backbone, 2 ′ position of nucleoside (nucleoside), terminal 3 ′ position, or the like. Suitable moieties for externally altering mass include, for example, halogen, azide, nitro, alkyl, allyl, sulfur, silver, gold, platinum, mercury, mass component types,
In the WR, the Wha chain group and R have a mass changing portion.

The linking group W is included in a covalent bond between a nucleotide or a nucleoside and R, and varies depending on the properties of the molecule. The functional groups typically present or introduced into the molecule are used for linkage. Linking groups vary from a bond to a chain with from 1 to 100 atoms, typically from about 1 to 60 atoms, preferably 1 to 4 atoms.
Up to 0 atoms, more preferably 1 to 20 atoms, typically carbon,
Each is independently selected from the group consisting of hydrogen, oxygen, sulfur, nitrogen, halogen and phosphorus. The atoms in the chain may be replaced with atoms other than hydrogen. As a general rule, the length of a particular linking group can be arbitrarily selected to be convenient for the synthesis and incorporation of the desired group. The linking group may be aliphatic or aromatic, although aromatic groups are usually included with the diazo group. The mass-changing R group can consist of the linking group itself, or can be attached to another mass-changing moiety by methods well known in the art. Common functionalities present in linking groups that form a covalent bond between the molecule to be attached and the mass changing moiety include alkylamines, amidines, thioamides, ethers, carbamates, ureas, thioureas, Includes guanidine, azo, thioether, and carboxylate, sulfonate, and phosphate esters, amides, and thioesters. For example, when an amine and a carboxylic acid, or a nitrogen derivative thereof or phosphoric acid are linked, amidine and phosphoric amide are formed. When the mercaptan and the activated olefin are linked, a thioether is formed. When the mercaptan and the alkylating agent are linked, a thioether is formed. When the aldehyde and the amine are linked under reducing conditions, an alkylamine is formed. When the carboxylic acid, phosphoric acid and alcohol are linked, an ether is formed.

For other suitable mass alterations, see Oligonuleotides and Analogues, A
Practical Approach, F. Eckstein (editor), IRL Press, Oxford (1991); including those disclosed in U.S. Pat. No. 5,605,798 and Japanese Patent No. 59-131909, will be apparent to those skilled in the art. And incorporated herein by reference.

A primary goal of the invention is to generate either complete mixtures or sets of mixtures for use in the mass range available for mass spectrometry, with the attendant goals having different base pairing patterns ( Sequence) to reduce mass overlap between oligonucleotides. As is apparent from the discussion herein, the amount and type of information sought in any analysis indicates the type of X-mer nucleotide required.

Next, three methods for synthesizing a mixture of X-mer nucleotide precursors will be described. This is not intended to be limiting, but merely as an example. Each of the methods described herein has advantages depending on the degree of control over the synthesis required for the particular oligonucleotide. All three methods utilize standard phosphoramidite chemistry or enzymatic reactions well known in the art. Different types of nucleotide precursor mixtures of varying mass contemplate the possibility of being synthesized for a given type of application. For example, defined mixtures that are easy to manufacture and inexpensive can be used in very high throughput, low resolution assays. More complex mixtures, which are more expensive to manufacture, can be prepared for higher resolution applications.

X-mer nucleotide precursors may be synthesized by conventional techniques, including methods using phosphoramidite chemistry, including both 5 'to 3' and 3 'to 5' synthetic routes. For example, to synthesize all of the hexamer nucleotides, 4
, 096 times of synthesis. The use of an automatic robot driving device facilitates this process. By this method, the synthesis of individual X-mer nucleotide precursors in terms of composition and length can be completely controlled. This is the maximum possible M
This is necessary to create a mixture of X-mer nucleotides with NC. The individual synthesis allows for QC analysis of each X-mer nucleotide and helps to make the final product. By making individual samples of each X-mer nucleotide, one can define a mixture of subsets to be made to increase compositional resolution. Furthermore, it allows each X-mer nucleotide to be at a particular concentration in the mixture. This may help to correct for differences in the expected thermal stability of each X-mer nucleotide / target duplex.

The X-mer nucleotide precursor is based on solid support phosphoramidite chemistry and A, C
, G and T phosphoramidites may be synthesized in parallel or alone using a 25% mixture series. For example, the synthesis involves the 3′-CPG-link 5
25% of each of the A, G, C and T nucleosides protected with '-DMT-
It is carried out stepwise starting from the mixture. For the synthesis of a total of 4,096 hexameric nucleotides, 25 for each of the A, G, C and T forms of phosphoramidite
% Bottles containing 5% mixture are prepared and used in each of the five condensation reactions. For example, the bottle for the first condensation reaction step contains phosphoramidite corresponding to 2'-O-methyl-2,6-diaminopurine, 2'-O-methylguanosine, 2'-deoxy-5-iodocytidine and thymidine. Included in 25% molar equivalent. The bottle for the second condensation reaction contains 2'-deoxyadenosine, 2'-
The phosphoramidite corresponding to deoxy-7-deazaguanosine, 2'-O-methyl-5 (1-propynyl) cytidine and 2'-deoxy-5-fluorouridine is contained in a molar equivalent of 25%. A 25% mixture of other types of modified A, G, C and T phosphoramidites has been made for the remaining three condensation steps.

Although this is a relatively simple method from a synthetic point of view, it imposes a mass interdependence on the resulting final X-mer nucleotide mixture. In the above example,
Of the hexamer nucleotides with a second A-type nucleotide, all 1,024 have a 2'-O-methyl-2,6-diaminopurine at that position. The third is G
Have all 7-deazaguanosine, and so on. Thus, although this synthetic scheme has reduced the overall ambiguity relative to the use of natural bases, the resulting positional interdependence in the mixture does not account for the final assay. Has a limit on the resolution detection power. This is evident from the MNC values calculated for PEA shown in Table 1. 6 of permutation combination
The MNC of the dimeric nucleotide mixture is significantly greater than that of the natural hexameric nucleotide mixture. However, it is about twice as low as an individually optimized set, which must of course be synthesized individually. It is important to note, however, that multiple permutational syntheses may be performed, such that a defined subset of X-mer nucleotides are each made in a separate synthesis. The products of the different syntheses are mixed together to make a complete mixture. Although there is still positional interdependence in any synthetic mixture, the combined mixture can have a greater mass complexity than the one-time permutation mixture described above.

In another method, oligonucleotides with altered mass can be synthesized individually as described in the first method, followed by chemical modification of the 5 'end with certain mass tag moieties. Only a few separate mass tags are needed to distribute the resulting natural oligonucleotide mixture over the mass range available for mass spectrometry. This method is similar to that disclosed in US Pat. No. 5,605,798, the relevant disclosure of which is hereby incorporated by reference. Although the aforementioned patent describes a similar synthesis, it does not describe the use of mass-tagged oligonucleotides in a characteristic type of analysis as described in the present invention, It should be noted that they have not suggested.

Effect of X-mer nucleotide modification on mass spectrometric, thermodynamic and enzymatic properties The composition of the X-mer nucleotide precursor directly affects the overall specificity and sensitivity of the assay. In addition, by controlling their design and their mode of synthesis, one can incorporate modifications that aid their use in the methods of the invention. For example, X
Internucleoside linkages may be modified in the phosphodiester backbone of the dimeric nucleotide. In one embodiment, it is preferred that such a chemical modification renders the phosphodiester bond resistant to nuclease degradation. Suitable modifications include the incorporated non-crosslinked thiophosphate backbone, 5'-N-phosphoramidite internucleotide linkages, and the like.

Altering the mass increases the thermodynamic stability of the hybrid formed between the X-mer nucleotide precursor and the target nucleic acid analyte and normalizes the hybrid's thermodynamic stability in the mixture there's a possibility that. For example, 2,6-diaminopurine forms a more stable base pair with thymidine than adenosine. In addition, incorporation of 2'-fluoro-thymidine increases the stability of AT base pairs, while incorporation of 5-bromocytidine and 5-methylcytidine increases the stability of GC base pairs.

[0130] The change in mass reduces the thermodynamic stability of the hybrid formed between the X-mer nucleotide precursor and the target nucleic acid analyte and normalizes the thermodynamic stability of the hybrid in the mixture. May be changed. AT base pairs can be destabilized by incorporating 2'-amino-nucleosides. Inosine can be used instead of guanosine for destabilized GC base pairs. N-4-
Incorporation of ethyl-2'-deoxycytidine has been shown to reduce the stability of GC base pairs. The latter incorporation can normalize the stability of any duplex if its stability holds independent of the amount of AT and GC (Nguyen et al., Nucleic Acids Res. 25, 3095 (1997)).

Modifications are also introduced to reduce fragmentation of the oligonucleotide due to the ionization process in mass spectrometry. For example, one method is a 7-deaza modification of purines that stabilizes N-glycoside linkages and thereby reduces fragmentation of the oligonucleotide during the ionization process (see, eg, Schneider and Chait, Nucleic Acids Res.
23, 1570 (1995)). Modification of the 2'-position of the ribose ring with an electron-withdrawing functional group such as a hydroxyl group or a fluoro group is used to reduce fragmentation by stabilizing the N-glycoside bond (see, for example, Tang et al., J. Am. Soc. Mass Sp
ectrom, 8, 218-224, 1997).

Mass Tagged Chain Terminating Nucleotides The use of chain terminating nucleoside triphosphates such as dideoxynucleoside triphosphates in the present invention for the PEA method is fundamentally well known in the art. Is different. "Scoring" hybridization between a target nucleic acid and a number of mass-modified X-mer nucleotides by shifting the mass of the resulting extension product among the mass-modified X-mer nucleotide precursors As a method, this PEA
The method utilizes a chain terminating nucleotide. This particular feature ensures that the absolute weight of the nucleotides terminating the chain is greater than the mass range defined by the lightest and heaviest X-mer nucleotide precursors in the mixture. For example, X-mer mass range of nucleotide precursors is composed of four natural deoxynucleotides made all 6-mer nucleotides, (C ₆₎ for from 1,667 atomic mass units (amu) (C ₆₎ Is 1,907 amu. The difference in mass range is now 240 amu. The mass of the natural dideoxynucleotide (monophosphate minus water molecule) is 296, 312, 272 and 287 amu for pddA, pddG, pddC and pddT, respectively. Therefore, the absolute mass of each dideoxynucleotide is greater than the mass range of the natural hexamer nucleotide mixture, which is sufficient to distinguish the mass of the X-mer nucleotide precursor from the X + 1 nucleotide extension product. However, for example, by changing the mass or using X-mer nucleotides of varying lengths,
When the mass range of the X-mer nucleotide precursor is increased, it is desirable to tag the chain terminating nucleotides with a mass so that any extension product has a greater mass than any X-mer nucleotide precursor.

In one embodiment of the present invention, the mass-tagged dideoxynucleoside triphosphate may have an additional chemical moiety. This enhances the ionization efficiency of the desired X-mer nucleotide relative to the unextended X-mer nucleotide precursor or undesired components present in the sample mixture. Typically, at least two factors increase ionization efficiency, but more typically are four factors, preferably ten. Thus, for example, for an X-mer nucleotide precursor, 6
When multimeric nucleotides are used, the additional components described above serve to facilitate the analysis of hexameric nucleotide precursors and heptameric nucleotide extension products. Examples of such additional chemical components are primarily amines, which act as protonation sites and support one cationic species for MALDI analysis (Tang et al., 1997
,the above). It is also possible to incorporate quaternary amines with a fixed positive charge.
As disclosed in Gut et al. In WO 96/27681, NHS ester chemistry may be used to incorporate such chemical groups into non-cleavable mass tags.
Briefly, succinimide esters of charged quaternary ammoniums, such as trimethylammonium hexylyl-N-hydroxysuccinimidyl ester, react with nucleoside derivatives having primary fatty acid amino groups. Suitable nucleosides are known terminators, such as, for example, 3'-amino derivatives of 2'-deoxynucleosides. Other suitable nucleosides include 5- [3-amino-1 such as those used to make the fluorescently labeled ddNTPs described by Prober et al., (Science 238, 336 (1987)). -Propynyl] -pyrimidine and 7-deaza [3-amino-1-propynyl] purine derivatives.

(Method of the Invention) (Preparation of Short Word Description in Target Nucleic Acid) The invention is based on the understanding of a target sequence in the form of a set of oligonucleotides (X-mer nucleotides) complementary to the target sequence, and mass spectrometry. Methods and reagents for analyzing sets in a method. The set of oligonucleotides represents the content of the target in "short words" and indicates defined sequence information about the target. The set of oligonucleotides representing the target is shown in three general types (FIG. 1). The nested set of overlapping X-mer nucleotides (FIG. 1a) is characterized by the long overlap of X-mer nucleotides in the set. The nested set of partially overlapping X-mer nucleotides (FIG. 1b) has less overlap of the X-mer nucleotides and the non-overlapping set of X-mer nucleotides (FIG. 1c) has no overlap. In the three types of sets, the length of the X-mer nucleotide in any set need not be constant. Generally, the X-mer nucleotides in the nested set of overlapping X-mer nucleotides are from about 3 to about 18 lengths, typically from about 5 to about 14 nucleotides. In this set, all but one nucleotide overlap in the entire length of the target nucleic acid sequence. Generally, X in a nested set of partially overlapping X-mer nucleotides
Multimeric nucleotides are about 3 to about 18 nucleotides in length, typically about 5 to about 14 nucleotides. Generally, the X-mer nucleotides in the nested set of non-overlapping X-mer nucleotides are from about 3 to about 18 in length, typically
From about 4 to about 14 nucleotides. For all three methods, X-mer nucleotides sample the full length of the target nucleotide sequence. The number of X-mer nucleotides actually created is generally determined by the length of the target nucleotide and the desired result. The number of X-mer nucleotides must be sufficient to achieve the stated application goals. For example, if the goal is to perform mutation detection, then a sufficient number of X-mer nucleotides are needed to distinguish between X-mer nucleotides or sets of X-mer nucleotides containing mutations.

(General Description of Method) One aspect of the present invention is a method for analyzing the sequence of a target nucleic acid. A mixture of the mixture of X-mer nucleotide precursors and the target nucleic acid sequence is allowed to form a hybrid. The mixture comprises natural and modified X-mer nucleotide precursors having a minimum length of 3 nucleotides. The mixture comprises at least 56 different X's
When containing a multimeric nucleotide precursor, the mixture has a mixture coverage complexity of about 7/8. As the average length of the X-mer nucleotide precursor increases,
The number of different X-mer nucleotide precursors in the mixture of the present invention may also increase, and the mixture coverage complexity may decrease. The minimum limit of the mixture coverage complexity is equal to 56 divided by the number of X-mer nucleotide precursors in the mixture. The length of the X-mer nucleotide precursor can be independently selected for each X-mer nucleotide precursor. The mass number complexity (MNC) in the mixture is higher than any natural equivalent mixture. The hybrid is processed to alter the mass of the X-mer nucleotide precursor portion of the hybrid in a target-mediated reaction, and the product is analyzed by mass spectrometry. The first two steps may be performed in solution or with nucleic acids bound to a surface such as an array. They are,
A solution-based system is preferred because it depends on the mass action and diffusion process of the standard solution.

Preparation of X-mer Nucleotide Mixtures The first step in the method of the invention is to provide natural and altered X-mers with appropriate mass numbers and coverage complexity for any application To prepare a mixture of nucleotide precursors. The mixture of X-mer nucleotide precursors comprises:
It also has the properties described herein for ionization and thermodynamic properties. X
The design and preparation of the mixture of multimeric nucleotide precursors may be performed as described herein.

Processing Step The second step in the method of the invention is to treat the hybrid to alter the mass of the X-mer nucleotide precursor portion of the hybrid as described herein. This change may be made by enzymatic or chemical reaction.
Suitable enzymatic techniques include polymerase extension assays, ligase assays, and the like. Suitable chemical techniques include condensation of activated X-mer nucleotide precursors with carbodiimides and cyanogen bromide derivatives. The following discussion is a brief description of the various processes. A more detailed discussion is set out below.

Polymerase Extension Assay For the polymerase extension assay (PEA), the amount of X hybridized by polymerizing one nucleotide at the 3 ′ end of the X-mer nucleotide precursor hybridized with a nucleotide polymerase. Extend the somatic nucleotide precursor (see FIG. 2). For one form of PEA, the polymerase extension and cleavage assay (PEACA), hybridization is achieved by polymerizing one or more nucleotides at the 3 'end of a hybridized X-mer nucleotide precursor using a nucleotide polymerase. The resulting X-mer nucleotide precursor is first extended and then shortened again by degrading the product with a 5 'to 3' exonuclease, endonuclease or chemical reagent depending on the specificity of the method performed (Figure 3 and 4).

Ligase Reaction For the X-mer nucleotide ligation assay (XLA), adjacent hybridized X-mer nucleotide precursors are ligated together by ligase prior to analysis (see FIG. 5). The X-mer nucleotide precursor is long enough to act as a good substrate for ligation by ligase, but not too long as a template for ligation of the complementary X-mer nucleotide precursor in the reaction mixture. Is preferred. It should be noted that linking all adjacent X-mer nucleotide precursors is preferred, but not a requirement.

The ligation assay may be performed on a surface-bound array (see FIG. 6). The array has a surface and a variety of oligonucleotide probes attached to it.
The probe comprises: (a) a cleavage linker attached to the surface; and (b) a nucleic acid sequence having a 3 ′ end and a terminal 5 ′ phosphate, wherein the nucleic acid sequence is 3 ′ of the nucleic acid sequence.
The terminus comprises a nucleic acid sequence attached to a cleavage linker. The method comprises: (1) forming a hybrid between the target nucleic acid sequence and the probe; (2) adding a mixture of X-mer nucleotide precursors to the target nucleic acid sequence; and (3) attaching the target nucleic acid thereto. Linking the hybridized X-mer nucleotide precursor adjacent to the 5'-terminal phosphate with a surface-bound probe to form a hybridized precursor / probe complex with the sequence; 4) cleaving the complex at the cleavage linker site; and (5) analyzing the complex in terms of the characteristics or set characteristics of each probe by mass spectrometry.

Detailed Description of the Method The following description is directed to three general methods for generating sets of oligonucleotides that indicate the content of the target in short words. Each method can produce one or more types of oligonucleotide sets depending on the reagents used. This description is illustrative and not intended to be limiting. As mentioned above, the first method is "
The second method is called the "Polymerase Extension Assay" (PEA) and the second method is called the Polymerase Extension and Cleavage Assay (PEACA). The third method is called the "X-mer nucleotide ligation assay" (XLA).

The basis of all methods is a mixture of oligonucleotides (X-mer nucleotides) composed of natural and / or altered mass nucleotides. It should be understood that different sets of mixtures can be designed to create different types of sets, and thus provide different amounts of target sequence information. By analyzing the mass peaks present in the mass spectra generated in the above method, by correlating these peaks with information about the X-mer nucleotide precursors in the mixture relevant to each mass spectrum, and the target sequence Prior information that may be related to the information determines the information sought from the target.

PEA is a common method for creating nested sets of overlapping or partially overlapping X-mer nucleotides. The method has three basic stages (FIG. 2). In the first step, a mixture of possible X-mer nucleotides, representing any possible X-mer nucleotide sequence or a subset thereof, hybridizes at random sites in the target nucleic acid sequence according to Watson-Crick base pairing rules. It can be so. In the second step, hybridization was performed using a mixture of one or more of a nucleotide polymerase such as a DNA- or RNA-dependent DNA polymerase and a chain-terminating nucleoside triphosphate such as dideoxynucleotide triphosphate (ddNTP). The X-mer nucleotide is extended by one nucleotide. In the third step, the resulting extended X-mer nucleotide, ie, X + 1 nucleotides, is analyzed by mass spectrometry.

The degree of overlap between the X + 1 nucleotide products depends on the sequence integrity of the X-mer nucleotide mixture being interrogated. For example, if all 4,096 hexamer nucleotides, and all four ddNTPs are present in the mixture, maximum overlap between the resulting heptamer nucleotides is possible. If there are only 4,096 possible subsets of hexamer nucleotides and / or a subset of 4 ddNTPs, there will be less overlap between heptamer nucleotides and gaps in sequence coverage.

It is important that the chain terminating nucleotides have sufficient mass to effectively separate the X-mer nucleotide precursor mixture from the X + 1 nucleotide extension product. It is preferred that all hybridized X-mer nucleotide precursors be extended, but it should be noted that this is not a requirement. In the present invention, the greater the number of hybridized X-mer nucleotide precursors extended, the higher the accuracy of the measurement.

The X-mer nucleotide hybridizes with the target nucleic acid and is extended in the presence of the chain-terminating nucleoside triphosphate by one nucleotide complementary to the target nucleotide adjacent to the hybridized X-mer nucleotide. On the condition that
Reagent combinations are restricted. Generally, an aqueous medium is used. Other ionizable co-solvents may be used. Typically, oxidized organic solvents of 1 to 6, more usually 1 to 4 carbon atoms, including alcohols, ethers and the like may be used. Typically, such co-solvents, if used, will be less than about 70 weight percent, and more typically will be less than about 30 weight percent.

[0147] The pH of the medium is typically in the range of about 4.5 to 9.5, and more typically is about 5.5.
To 8.5, preferably about 6 to 8. Various buffers may be used to achieve the desired pH and to maintain the pH during the measurement. Buffers that may be mentioned by way of example include boric acid, phosphoric acid, carbonic acid, Tris, barbital and the like. The use of a particular buffer is not critical to the invention, but in a particular method one buffer may be preferred over another.

The reaction involving the chain terminating nucleoside triphosphate is performed for a time sufficient to produce an extended X + 1 nucleotides. Generally, the time to perform the entire method is about 10 to 200 minutes. It is usually desirable to minimize time.

The concentration of nucleotide polymerase is usually determined by empirical validity. Preferably, it is used at a concentration sufficient to extend most, if not all, of the X-mer nucleotide precursor specifically hybridized to the target nucleic acid (see below). The main limiting factors are generally reaction time and reagent cost.

The number of target nucleic acid molecules can be as low as 10 ^{5 in} a sample, but can generally vary from about 10 ⁶ to 10 ¹³ , and more typically from 10 ⁸ to 10 ¹² molecules in the sample. Preferably it is at least 10 ^-13 M in the sample, and may be between 10 ^-13 and 10 ^-6 M, and more usually between 10 ^-11 and 10 M ^-7 . Generally, the reagents for the reaction are provided in an amount that achieves extension of the hybridized X-mer nucleotide. The number of molecules of each X-mer nucleotide precursor is generally 10 ¹⁰ for a sample size of about 10 microliters, typically about 10 ¹⁰ to about 10 ¹³ , preferably about 1
0 ¹¹ to about 10 ¹² . The concentration of each X-mer nucleotide precursor is adjusted according to its thermal stability as discussed above. The absolute ratio of target to X-mer nucleotide precursor should be determined empirically. The concentration of the chain-terminated nucleoside triphosphate in the medium can vary depending on the affinity of the nucleoside triphosphate for the polymerase. Preferably, the reagent is present in excess. The nucleoside triphosphate is typically present at about ^10-7 to about ^10-4 M, preferably about ^10-6 to about 10M- ⁵ .

The reaction temperature will vary from about 0 ° C. to about 95 ° C., depending on the type of polymerase used, the concentration of target and X-mer nucleotides, and the thermodynamic properties of the X-mer nucleotides in the mixture.
Can be For example, under the conditions of 40 nM target nucleic acid sequence, 40 nM hexamer nucleotides and 7 nM Bst polymerase, the sequence of hexamer nucleotides allows 20% to 50% of hexamer nucleotides to elongate at 5 ° C. for 2 hours. Is done. The similar extension efficiency obtained at 20 ° C. indicates that extension efficiency is not solely due to thermodynamics in the X-mer nucleotide / target interaction. Importantly, it may be advantageous to have a cycle of the incubation temperature. By having a cycle, the structured region of the target may be exposed to the binding portion of the X-mer nucleotide, helping subsequent extension and promoting production of extension products. Therefore, if any target molecule has more than one X
It can act as a template in conjugation of monomeric nucleotides and subsequent extension reactions, and the overall sensitivity of PEA can be significantly increased. According to this aspect of the invention, one cycle may be performed at a temperature of about 75 ° C. to 95 ° C. for about 0.1 to 5 minutes, and more usually about 0.5 to 2 minutes, and another cycle may be performed at a temperature of about 5 ° C. to 95 ° C. It may be performed at 45 ° C. for about 1 to 20 minutes, more usually about 5 to 15 minutes. The number of cycles may be from about 2 to 20 or more. In general, the temperature and time of the cycle are selected to optimize extension on hybridized X-mer nucleotides of any length.

The order of mixing the various reagents forming the combination may vary. Typically, a sample containing the target nucleotide is mixed with a previously prepared chain-terminating nucleoside triphosphate and a nucleotide polymerase. X-mer nucleotides may be included in the pre-prepared mixture or subsequently added separately. However, adding all of the above simultaneously and adding step by step or step by step may be performed on condition that all of the above-mentioned reagents are mixed before the start of the reaction.

PEACA is another common method of creating nested sets of overlapping and partially overlapping X-mer nucleotides. There are four basic steps in this method (FIG. 3). In step one, every possible X
A mixture of X-mer nucleotides representing a multimeric nucleotide sequence, or a subset thereof, is allowed to hybridize at random sites in the target nucleic acid sequence according to Watson-Crick base pairing rules. In step 2, the nucleotide polymerase and one or more natural nucleotide triphosphates (dNTPs)
Alternatively, hybridized X-mer nucleotides are extended using dideoxynucleotides (ddNTPs) of altered mass. In step 3, the 5'-terminal portion of the X-mer nucleotide extension product is cleaved by enzymatic or chemical cleavage. In step 4, the resulting X-mer nucleotide product is analyzed by mass spectrometry.

PEACA is designed to create extension products of X-mer nucleotides, the length of the product being defined by the length of the target and the type of dNPT and the chain terminating nucleotide (ie, ddNTP) in the reaction mixture. . The potential overlap between the X-mer nucleotide products depends not only on the length, but also on the coverage complexity of the X-mer nucleotide precursor mixture. Using X-mer nucleotide precursors with greater coverage complexity will result in greater overlap between extension products. If all four dNTPs and one chain terminating nucleotide (ie, ddNTP) are in the reaction mixture, dNTP / d
Increasing the molecular ratio of dNTPs not only increases the length of the extension products, but also increases the likelihood of overlap between the extension products. Conversely, the smaller the dNTP / ddNTP, the shorter the extension product and the fewer potential overlaps.

In PEACA (FIG. 3), cleavage at some site at the 5 ′ end is defined by designated nucleotides in the X-mer nucleotide precursor. PEACA has the additional advantage of using enzymatic or chemical treatments to remove portions of the 5 'end of the X-mer nucleotide precursor. Such a property is exploited to make PEACA products of the same length when starting with a mixture of X-mer nucleotides of different lengths. This makes it possible to produce X-mer nucleotides rich in A / T that are longer than those rich in C / G (to correct for differences in thermostability), and to be the same or similar by cleavage. PEACA products of length can be made. In addition, the 5 'end of the X-mer nucleotide tends to hybridize incorrectly to the target and is not sensitive to polymerase. Therefore, the PEACA reaction may be used to remove incorrect information prior to mass spectrometry.

In the second type of PEACA (PEACA II, FIG. 4), it is incorporated during the extension process and cleavage is defined by the specified nucleotide. In this form, no portion of the X-mer nucleotide precursor remains in the X-mer nucleotide end product.
In addition to depending on which dNTPs and ddNTPs are present in the reaction mixture, if both optional nucleotides are present in the reaction mixture, dNTP / ddNTPs
The product length can also be determined by changing the molar ratio of For example, dNT
The longer the P / ddNTP ratio, the longer the extension product on average. Conversely, dNT
The smaller the P / ddNTP ratio, the shorter the extension product.

The conditions for performing the PEACA extension reaction are similar to those described above for PEA.
Buffer pH, ionic strength, surfactant addition, temperature (and its cycle),
Polymerase concentration, X-mer nucleotide concentration, target concentration, and dNTPs
All / ddNTP concentrations and ratios are optimized for maximum specificity, extension efficiency and information content.

The following illustration of truncating certain portions of the 5 ′ end is illustrative and not limiting, and will be described next. In one method, nucleotides determined by cleavage may be used. The nucleotide determined by cleavage can be, for example, a ribonucleotide that is sensitive to cleavage by an endonuclease such as ribonuclease A, or by a chemical base such as ammonia or hydroxide. The nucleotide determined by the cleavage can be, for example, a nucleotide that forms a 5′-N-phosphoramidite internucleotide linkage that can be cleaved by an acid such as trichloroacetic acid or dilute hydrochloric acid. When the X-mer nucleotide is formed only of ribonucleotides, for example, a deoxyribonuclease such as DNase I can be used.

The breakpoint can also be determined by nucleotides or sets of nucleotides that prevent cleavage by defined reagents or enzymes. For example, the X-mer nucleotide is 3
It can be composed of a triphosphate linkage at the 'end and a natural phosphodiester linkage at the 5' end. Cleavage of the X-mer nucleotide by a 5 'to 3' endonuclease that is sensitive to triphosphates, such as the T7 gene 6 protein, degrades the X-mer nucleotide to the point of the triphosphate linkage.

Conditions for performing the cleavage are those generally known in the art for the above enzymes. Briefly, such a condition is to incubate the enzyme in a suitable buffer containing divalent metal ions (if necessary). Suitable 5 'to 3' endonucleases, ie enzymes which cleave one nucleotide at a time from the end of the 5 'to 3' polynucleotide are, for example, DNA polymerase and T17 gene 6. . Suitable endonucleases, ie enzymes that cleave bonds in nucleic acids, are, for example, deoxyribonuclease I, ribonuclease A and the like. Chemical reactions suitable for chemical degradation are, for example, reagents that catalyze the hydrolysis of phosphate esters by acid or base and such as, for example, hydrochloric acid, trichloroacetic acid and the like. Conditions for conducting the above enzymatic or chemical reactions are well known to those skilled in the art and will not be repeated here.

XLA is another common method of creating a set of overlapping and half-overlapping X-mer nucleotides. The method has three basic steps (FIG. 5). In step 1, a mixture of X-mer nucleotides representing all possible X-mer nucleotide sequences, or a subset thereof, is capable of hybridizing at random sites in the target nucleic acid sequence according to Watson-Crick base pairing rules. To Step 2 hybridizes adjacent to each other using a ligase, such as DNA ligase, to help form a phosphodiester bond to join two adjacent bases on separate oligonucleotides. X-mer nucleotides are linked enzymatically. Such ligases include, for example, T4 DNA ligase, Taq ligase, E. coli ligase, and the like. Alternatively, adjacent X-mer nucleotide precursors may be chemically linked using a condensing agent. Suitable condensing agents include, for example, carbodiimides, cyanogen bromide derivatives, and the like. In step 3, the ligated X-mer nucleotide product is analyzed by mass spectrometry.

The degree of overlap between the n X-mer nucleotides depends on the completeness of the sequence of the X-mer nucleotide mixture under investigation. For example, if all 4,096 hexameric nucleotides are present in the mixture under investigation, the resulting n
Maximum overlap between species X-mer nucleotides is possible. If there are only 4,096 possible subsets of hexamer nucleotides, there will be less overlap between the n Xmer nucleotides.

The conditions for carrying out the reaction in this method are similar to the conditions described above. The pH of the medium is typically in the range of about 4.5 to 9.5, more usually in the range of about 5.5 to 8.5, and preferably in the range of about 6 to 8.

The reaction is performed for a time sufficient to produce the desired ligation product. Generally, the time required to perform the entire method is from about 10 minutes to 200 minutes. It is usually desirable to minimize time.

The reaction temperature will vary from about 0 ° C. to about 95 ° C., depending on the type of polymerase used, the concentration of target and X-mer nucleotides, and the thermodynamic properties of the X-mer nucleotides in the mixture.
Can be As in the case of PEA and PEACA, it may be advantageous to have a cycle of the incubation temperature. Cycling exposes the structured region of the target to the binding portion of the X-mer nucleotide,
It may assist in subsequent extension and promote production of extension products.

[0166] The concentration of ligase is usually determined by empirical validity. Preferably, it is used at a concentration sufficient to bind most, if not all, of the X-mer nucleotides specifically hybridized to the target nucleic acid. The main limiting factors are generally reaction time and reagent cost.

The concentration of each X-mer nucleotide precursor is generally as described above for PEA and may be adjusted according to thermal stability as discussed above. The absolute ratio of target to X-mer nucleotide precursor is determined empirically.

The level of phosphorylation at the 5 'end of a mixture of X-mer nucleotides can affect the degree of ligation (total number of ligation products) and the length of ligation products (n-value). The degree and length of ligation can also be controlled by introducing modifications that prevent ligation at the 3 'end of the X-mer nucleotide. In one method, two sets of X-mer nucleotide mixtures are put together in one reaction mixture. The X-mer nucleotides in the first X-mer nucleotide mixture have a 5 ′ phosphorylated end and a 3′-blocked end (py), and the X-mer nucleotides in the second X-mer nucleotide mixture are:
Both 5 'and 3' have hydroxy ends (oo). This gives oo / p
Only two X-mer nucleotide ligation products forming -y result. Blocks at the 3 'end may be non-condensable groups, such as non-natural groups such as 3'-phosphate groups, 3'-terminal dideoxy groups, polymers or surfaces, or other means of preventing binding. Can be performed. This method has significant information advantages because the two sets can be optimized together.

The use of modified X-mer nucleotides to control the extent and length of ligation
It can be combined with incorporation of ionization labels to increase ionization efficiency. The X-mer nucleotides of the initial X-mer nucleotide mixture have a 5 'phosphorylated end and a blocked, labeled 3' end (p-z). The X-mer nucleotides in the second X-mer nucleotide mixture are both 5 'and 3' hydroxyl-terminal (oo). This allows the two X-mer nucleotides to be oo
Only ligation products forming / pz are produced. The group represented by z can be a single functional group that serves two purposes, both as a block and an ionization tag, such as a quaternary ammonium group attached through a 3 'hydroxyl group, and can be attached to a nucleoside base. It can also be represented by a separate functionality, such as a 3 'dideoxy group for blocking, with another ionization tag attached.

[0170] PEA, PEACA and XLA have a number of desirable properties. First, they are all solution-based systems, which depend on the mass action and diffusion process of the standard solution. This is in contrast to hybridization systems on surface-based arrays, where the probes are physically attached to the surface, cannot diffuse, and the kinetics of hybridization are reduced. Become slow. For surface-bound arrays, the invention features a great variety of oligonucleotides that bind to target sequences. This may increase the overall efficiency of X-mer nucleotide binding and subsequent enzymatic reactions. Furthermore, X
Because the monomeric nucleotide precursors are short, they are less likely to form intramolecular structures.

Second, PEA, PEACA and XLA are advantageous for highly specific enzyme reactions. In the case of PEA and PEACA, the high specificity for a perfect duplex is essentially due to the hybridization process by extending only those primers that are hybridizing to the correct target sequence (and thus contributing to detection). `` Calibration (pro
of reading). This “proofreading” may increase the overall specificity when assaying those obtained by the hybridization method performed alone. Hybridization efficiency and specificity may also be enhanced by the ligase enzyme in XLA.

Third, unlike surface-based array hybridization systems that rely on the detection of hybridization itself, PEA, PEACA and XLA can detect even transiently stable primer-target interactions. . The time of interaction between the X-mer nucleotide precursor and the target may be long enough for the polymerase or ligase to recognize and act. This allows any target sequence to act as a template for binding of multiple precursors and subsequent extension or ligation reactions. This ability to detect cycling and transient events can increase the overall detection sensitivity of the method to that obtained in surface-based hybridization assays. As discussed above, this type of reaction cycling
Cycling the temperature during the extension or ligation reaction can be significantly facilitated.

Finally, the extension or ligation products generated by the method have a larger mass range than the X-mer nucleotide precursor. Thus, the spectral peaks resulting from unreacted precursor should not interfere with the mass spectral characteristics of the desired extension or ligation product. It is also contemplated that in the case of PEA and PEACA, the possibility of utilizing mass-tagged ddNTPs. This allows for greater assay flexibility and multiplexing of mass analysis steps.
The incorporation of an ionization tag into the X-mer nucleotide precursor via a 5 'or 3' linker, directly into a base or sugar or chain terminating nucleotide, is also contemplated. Such properties help to increase the overall sensitivity of the assay, simplify the separation step, and eliminate it if possible, and facilitate assay automation and sample throughput.

The first three methods described here are directed to freeing the target under investigation in solution. However, XLA methodology can be used in conjunction with surface-bound oligonucleotides as an array of oligonucleotides to increase the overall resolution power in the array system. Arrays generally include a surface consisting of a mosaic of various oligonucleotides, the oligonucleotides being discontinuous on the surface, individually lining up in known areas. Such ordered arrays containing large amounts of oligonucleotides have been developed as tools for high-throughput analysis of genotype and gene expression. Oligonucleotides synthesized on the solid support recognize unique nucleic acids by hybridization, define specific target sequences, analyze gene expression patterns, or identify specific gene mutations The array can be designed to

The present invention may be practiced with oligonucleotides attached to a support.
Referring to FIG. 6, according to the present invention, an oligonucleotide array such as a DNA array is formed so that a DNA probe is attached to the surface at the 3 ′ end via a kind of photocleavable or chemically cleavable linker. can do. Linkers can be cleaved by light, chemical, oxidation, reduction, acid labile, base labile and enzymatic methods. Probes bound to such a surface also have a phosphate at the 5 'end. An example of a photocleavable linker is as based on the o-nitrobenzyl group as described in WO95 / 04160 and the like. Examples of linkers that can be cleaved by reduction include those with a dithioate functionality that is cleaved by mild reducing agents such as dithiothreitol or β-mercaptoethanol. Examples of unstable and cleavable linkers include those containing 5'-N-phosphoramidite internucleotide linkages or nucleotides as components of the linker. Linkers that can be cleaved by base instability include those that contain ribonucleotides as a component of the linker.

Referring to FIG. 6, an array-based X-mer nucleotide ligation assay (A
XLA) includes four steps. In step 1, the target sample hybridizes with the probe bound to the surface of the array under conditions compatible with the ligation reaction described above. The target nucleic acid is unlabeled or labeled with a fluorescent label or the like. In step 2, a mixture of X-mer nucleotides (not phosphorylated) is added to the array and allowed to hybridize randomly with the target according to Watson-Crick base pairing rules. In step 3, the X-mer nucleotide hybridized adjacent to the oligonucleotide bound to the surface is DNated as described above.
Ligation using A ligase. Since only oligonucleotides attached to the surface have terminal 5 'phosphates, ligation only occurs between hexamer nucleotides and X-mer nucleotides that form a hybrid adjacent to the DNA probe and are adjacent to each other. No ligation takes place between hexamer nucleotides Xmer nucleotides in tandem or hybridizing to other parts of the target. In step 4, the linked X-mer nucleotide is cleaved from the surface and analyzed for each feature (or set of features) using mass spectrometry. The conditions for performing the ligation reaction in this method are similar to the conditions described above.

The cleavage step of the oligonucleotide and the ionization with the aid of the matrix may be performed simultaneously. This requires a system that is compatible between the conditions required for hybridization, photocleavage of oligonucleotides, and matrix formation and ionization, but requires cleavage from reaction vessels such as microtiter dishes to others. There is no need to transfer the produced product. In addition, the likelihood that the ligation product will diffuse from one feature to another after the photocleavage step prior to analysis can be minimized.

In the above format, the mass number complexity (MNC, see discussion below) is multiplied by the actual feature number of the array, the MNC of the X-mer nucleotide precursor (see discussion below). Become. In this situation, the hexameric nucleotide oligonucleotide (4,4
096 Features) Making all arrays and performing XLA with a mixture of X-mer nucleotides yields a sufficient number of total effective features for new sequences larger than 1 kB DNA fragment. This method can also be combined with a non-uniform array structure to further increase the information content of the array. Such non-uniform array structures include, but are not limited to, a universal base or a pair that can be paired with all four natural bases at a defined site in the X-mer nucleotide, as an example. Includes the incorporation of either modified base that pairs with a limited portion of the natural base. The features of this type of structure have been described for surface-based hybridization array systems (Pevzner PA, et al., J. Biomolecular Structure Dynamics 9, 399).
(1991)).

Design of Precursor X-mer Nucleotide Mixture The power in each type of assay described above depends on the properties of the X-mer nucleotide mixture used to probe the target nucleic acid. As discussed above, the high degree of mass overlap between X-mer nucleotides with different sequences is an unavoidable conclusion of an X-mer nucleotide composed of only four building units (FIG. 7a). Histogram). The reagents of the present invention are designed to reduce ambiguity and thus increase power in all applications utilizing mass spectrometry to analyze the sequence of a target nucleic acid. High mass number complexity (MNC (Ω)
) And mass-modified X with coverage complexity (CC (Ω))
This reduction can be achieved by using a mixture of monomeric nucleotide precursors (Ω). Furthermore, the mixtures of the invention are general and universal in the sense that they may be used in any application that has the goal of determining the sequence information of the target nucleic acid. In addition, mixtures may be designed without reference to prior information regarding the target nucleic acid sequence, for example, including the presence, location, or identity of the mutation.
However, this does not mean that the mixture is not useful for analyzing a target nucleic acid sequence for which some information is known in advance,
It does not mean that prior information is not useful for the interpretation of the mass spectrum.

For specific applications (eg, mutation detection), the power of the assay is
It is measured with a target nucleic acid of such length that the problem is solved at any success rate, eg 95% (detection of the mutation in this particular example). As the power of the assay increases, so does the length that can be analyzed at any success rate. The same applies to the success rate at which any length can be analyzed. A good criterion for ease of use is the length of DNA that can be analyzed with an automated DNA gel electrophoresis sequencer, about 500 bases. A reasonable goal is to analyze a 500 base target with a> 95% success rate.

To determine the theoretical power of the assay, simulate the mass spectrum of X-mer nucleotides for a typical target in a randomly drawn DNA sequence or genomic DNA drawn from a genomic database, It is enough to see if the desired information can be extracted from the results. However, this is a time consuming process, especially when many alternative assay designs are considered. Therefore, a proxy has been set up to replace assay power measurement, as described below. The assay is optimized using this proxy. Final analysis is performed via simulation of the assay.

The measure used for optimization is the average ambiguity (A (Ω)) defined above. (A (Ω)) is given by calculating the mass histogram of potential products of X-mer nucleotides in each reaction mixture and then calculating the average number of products that can be confused with each individual product. If the product length is N and the histogram for mixture i has hi (m) masses m (ie, the mass of possible hi (m) products in this mixture is m), The average ambiguity is the sum i (total m (hi (m) ² / (4 ^N ))). The optimization process can be considered as an attempt to make the histogram as wide and flat as possible (FIGS. 7b and 7c).

If a complete determination of the content by short words is desired, it should be noted here that the histogram should not have more than one peak. In this case, the optimization protocol should reduce the height of the largest peak in the histogram, rather than reduce (A (Ω)). By selecting one oligonucleotide from each mass and placing it in a separate mixture, the mixture required for complete short word analysis is created. This allows specific masses to be detected in the analysis of mixtures that are clearly related to the selected X-mer nucleotide. Thus, the height of the largest peak in the final histogram determines the number of mixtures.

Mass number complexity (MNC), a measure of the power of the assay, is determined by dividing (A (Ω)) by the total number of potential products. This is a useful measurement when comparing assays where the number of products is different. According to our simulations, the MNC calculated in this way can be used to detect mutations, identify the type and location of mutations, and determine the genotype of a single nucleic acid polymorphism. It was found that it was monotonically related to the target power. Therefore,
It is reasonable to optimize with this proxy. For reference, ambiguity is always one for arrays with probes bound to the surface for all oligonucleotides of n-mer nucleotides, and the number of valid features is equal to the number of oligonucleotide probes.

As mentioned above, the main goal of the design process is to define the average ambiguity (A (
Ω)). If only natural bases are used in one reaction mixture of hexameric nucleotides, the ambiguity and MNC are fixed using the formula given above (
(MNC = 53), equivalent to the information obtained with a homogeneous fragment of the target. Modified bases such as 5-iodocytidine, 5-fluorouridine and 2'-O-methylguanosine can be used to disambiguate oligonucleotides by selectively replacing certain bases by modification. it can. For example,
When a natural base is indicated by ACGT and a modified set is indicated by A * G * C * T *, the oligonucleotide A * TATATT can be distinguished by the mass from the oligonucleotide TATATAT. Thus, optimization involves selecting the appropriate substitutions, limiting the available phosphoramidite bases, selecting the synthesis strategy, the extent of ligation or extension, the length of the precursor oligonucleotide, and the number of reaction mixtures. It is the process of doing.

FIG. 8 illustrates the optimization process for PEA when the X-mer nucleotide precursor X-mer nucleotides are individually synthesized into hexamer nucleotides. In the process, the X-mer nucleotide is extended by one nucleotide using the natural dideoxynucleotide, the chain-terminating nucleoside triphosphate, to repeat the target with a short word of the 7-mer nucleotide using one reaction mixture. I do. The optimization process is directed at creating a complete set of hexamer nucleotide precursors so that the resulting histogram of the 7-mer nucleotide product is wide and flat.

To illustrate, the algorithm of FIG. 8 includes two adenylate derivatives (A),
That is, 2'-deoxyadenosine and 2'-O-methyl-2,6-diaminopurine, having two masses of 330 and 375, respectively, and two cytidylic acid derivatives (C), ie, having masses of 306 and 434, respectively. 2'-deoxycytidine and 2-deoxy-5-iodocytidine, two guanylic acid derivatives (G
), Ie, 2′-deoxyguanosine and 2′-O-methylguanosine with masses of 346 and 376, respectively, and two thymidylate derivatives (T)
I.e., 2'-deoxythymidine and 2'-deoxy-5-iodouridine having masses of 321 and 433, respectively.

Start by considering a mixture of all-natural hexameric nucleotides. Then, a mass histogram of the mixture is calculated, and the mass is classified according to the peak size (frequency). X-mer nucleotides are selected from the peak with the highest frequency (maximum number of hexamer nucleotides at any mass), and an alternative mass is calculated for such X-mer nucleotides using the reagents described above. The X-mer nucleotide is then redistributed to the alternative mass with the lowest frequency in the mass histogram, provided that this re-allocation results in a flat histogram. If none of the X-mer nucleotides has a high peak that can be reassigned, then the X-mer nucleotide is selected from the smaller peak and examined. Once the X-mer nucleotides have been successfully located,
Once reassigned, the complete mass histogram is calculated again and the process is repeated according to the criteria described until there are no more X-mer nucleotides available for reassignment.

As a result of the above optimization process, an average ambiguity of less than 30 is observed for the molecules used. This is calculated by applying the above average ambiguity equation to the final mass histograms in FIGS. 7b and c. In this case, each peak of the mass histogram contains about 30 out of a total of 16,384 possible heptamer nucleotides (FIGS. 7b and c). This gives an MNC of about 559, as shown earlier and calculated by the above equation (see Table I).

According to simulations, the value for MNC is about 0 with a 95% success rate
. This corresponds to the ability to detect one point mutation in a 9 kB single-stranded DNA (Table I). In this sense, such a mixture can be said to provide information equivalent to a hybridization array bound to a surface with 559 features. An advantage of the present invention is that PEA assays are common, while arrays are not, in practical applications. This should indicate that with the PEA described above and hexamer nucleotides, point mutations can be examined for any target sequence of 0.9 kB with an average success rate of 95%.

For PEA, splitting the product into separate reaction mixtures will lead to reduced ambiguity depending on the approximate number of mixtures used. One particularly simple method is as follows. Make four different reaction mixtures. Each mixture contains all of the X-mer nucleotide precursors and only one of the four natural dideoxynucleotide terminators. The four mass spectral peaks are absolutely assigned to the terminus of the 7-mer nucleotide in the particular extension. In this way, the MNC is increased by one of the values from 4 to 2,075. Mutations in the 1.6 kB double-stranded heterozygous PCR product can be detected with this value of MNC (see Table I). The same effect can be obtained by using four mass-tagged dideoxynucleotide terminators in one reaction (see FIG. 9). In this case, the mass tag is designed so that the mass spectra of the extended 7-mer nucleotide product are separately captured according to the type of the dideoxynucleotide at the 3 ′ end.

Mass ambiguity may also be reduced by “binning”, ie, examining the target by individual subsets of the mixture and combining the results of each during the analysis. The mixture may be binned by partitioning such that each individual X-mer nucleotide is in one of the partitioned mixtures.
Alternatively, the mixture may be binned such that any X-mer nucleotide enters more than one partitioned mixture.

The mixtures or sectioned mixtures of the invention may be designed to reduce ambiguity in the mass spectrum of the target nucleic acid sequence to any desired level. The level of ambiguity may be reduced to a minimum level so that the full content of the short word of the target nucleic acid sequence can be determined.

In addition to mass number complexity, the resolution power and generality of PEA depends on the coverage complexity (CC (Ω)) of the X-mer nucleotide mixture. Simulations have shown that X-mer nucleotide mixtures having significantly less than the theoretical maximum CC (Ω) have sufficient resolution power and generality. As shown in FIG. 10, 50% of the total 4,096 species in hexamer nucleotides are optimally capable of detecting one mutation at any success rate without significantly affecting target length. From the hexamerized hexamerized nucleotide mixture. As the X-mer nucleotides are further removed from the mixture, the power of the assay decreases rapidly. This effect is an equilibrium that is established between increasing the total number of X-mer nucleotides to maintain coverage generality, but decreasing the total number of X-mer nucleotides to reduce mass overlap. It is due to. As expected, a high success rate requires a high ratio of total X-mer nucleotides. Probing the target with two separate mixtures, commonly optimized to have orthogonal information, is expected to yield a very high success rate (> 99%).

For XLA as well, the power of the assay is dependent on the MNC and CC (Ω) of the X-mer nucleotide precursor mixture. As shown in Table I, XLA using a complete hexameric nucleotide mixture of sequences composed of natural nucleotides resulted in approximately 2
An MNC of 00 is obtained. However, when performing XLA with a modified hexamer nucleotide mixture that has been optimized for PEA, the MNC increases by a factor of 2 to about 400. It should be noted that the characteristics of the optimized histogram for the X-mer nucleotide precursor are different for PEA and XLA. In general, while the histogram of the X-mer nucleotides optimized for PEA is flat, the histogram of the X-mer nucleotides optimized for XLA is bimodal with a bias toward the end of the mass range. This produces a product histogram in which the ligation effect of the X-mer nucleotide precursor is centered in the middle of the mass range,
This is because the MNC is lowered. To raise the MNC against this trend, it is helpful to create a histogram of the X-mer nucleotide precursor with a bias at the low and high mass boundaries.

As in the case of PEA, complex MNCs for XLA can be enhanced by examining a defined subset target of a diverse mixture of X-mer nucleotides. However, for XLA, optimized binning of the X-mer nucleotide precursor is problematic, as each precursor must have the opportunity to link with any other precursor to maintain the generality of the assay. Thus, if an S-set of X-mer nucleotide precursors are made and the assay reaction connects X-mer nucleotide pairs, an S (S-1) / 2 reaction mixture will be created.

As will be apparent from the above discussion, the precise optimization process will depend on the type of assay and the details of the information sought. In one example, an exhaustive list of all X-mer nucleotide precursors can be made and the best one selected. For example, using the permutational combinatorial strategy described for the hexamer nucleotide precursor below, this strategy is realistic. That is, the number of potential precursor sets is 2 ^24/6 ! This is a number that can be thoroughly analyzed in a reasonable time. In the more complicated case, an optimization algorithm like that described for PEA is used.

A difficulty that can arise is the presence of isotopic peaks in the spectrum that result from the isotopic composition of the molecule. It means the amount of a particular isotope for each atom in the molecule. For example, natural carbon is 99% carbon 12 and 1
% Carbon-13. An oligonucleotide consisting of a representative heptamer nucleotide has about 70 carbon atoms and gives rise to two almost as strong peaks at different atomic mass units. One way to address this problem is to rely on high performance data analysis methods with noise processing of isotopic peaks.
This uses natural isotope yields to calculate and / or empirically determine the true mass distribution of potential X-mer nucleotide extension products. This information is then used to denoise the overlap between the isotopic peaks of the various X-mer nucleotides. Another approach to addressing this problem is to partition the X-mer nucleotide precursors into a mixture of subsets, with one subset containing only even pure elongation or ligation major masses and the other subset containing Only the odd major mass is included. Thus, the N + 1 dominant isotope peak (primarily due to carbon 13) cannot interfere with the other dominant peak, because the dominant mass is not created in any subset mixture. Alternatively, purified carbon 12 may be used to make an X-mer nucleotide precursor. In this method, the basic nucleoside components can be isolated from bacteria or yeast cultured in a culture solution containing a purified or enriched nucleoside precursor or carbon source containing carbon 12 (eg, Crain, PF, Meth
ods Enzymol. 193, 782 (1990), Nikonowica, DP et al., Nucleic Acids Res.
20, 4501 (1992), Hall, KB, Methods Enzymol. 261, 542 (1995), Macallan
Natl. Acad. Sci. USA 95, 708 (1998)). The nucleoside is then converted to the appropriate phosphoramidite for automated synthesis using standard protocols (eg, oligonucleotides and analogs, the practical method F. Eckstein (ed.
itor) IRL Press, Oxford (1991)). A similar method may be used with isotopic peaks introduced by using altered nucleotides or ionization tagged nucleotides. Mass tags or ionization tags can be prepared from isotope enriched precursors, or, alternatively, some or all of the naturally occurring elements as one isotope, such as fluorine, phosphorus or iodine. All may be configured.

(Analysis Step) The reaction mixture or the purified X-mer nucleotide product is subsequently analyzed by mass spectrometry. The details of the analysis are well known in the art and will not be repeated here. Suitable mass spectrometry methods are described in Methods in Enzymology B. Karger & W. Hancock (ed
itor), Academic Press, San Diego, V270 (1996) and Method J in Enzymology.
McCloskey (editor), Academic Press, San Diego, V193 (1990). These include matrix-assisted laser desorption / ionization (MALDI),
Electrospray ("ESI") ion cyclotron resonance ("ICR"),
Includes Fourier transform and delayed ion extraction and combinations and variants above.
Suitable mass analyzers include magnetic, sector / magnetic deflection devices in single quadrupole, ternary quadrupole (MS / MS), Fourier transform and time-of-flight ("TOF") configurations.

It is also contemplated that the reaction products be purified prior to mass spectral analysis using techniques such as, for example, high performance liquid chromatography (HPLC), capillary electrophoresis, etc. (see FIG. 5). Reverse phase HPLC may be used to separate the extended or ligated X-mer nucleotide products based on hydrophobicity. The resulting HPLC fraction is analyzed by mass spectrometry. Such techniques may significantly increase the resolution power in the claimed method.

For analysis by MALDI or the like, it may be desirable to modify the X-mer nucleotide precursor or X-mer nucleotide to convey desired characteristics to the analysis. Examples of such modifications include those done to reduce laser energy, volatilizing X-mer nucleotides, minimizing fragmentation, predominating single charged ions, To normalize the reaction of the desired oligonucleotide, regardless of its sequence or sequence, to reduce the peak width, and to increase the sensitivity and / or selectivity of the desired analysis product. For example, modification of the phosphodiester backbone by cation exchange helps to eliminate peak broadening due to heterogeneity of the bound cations per nucleotide unit. Alternatively,
X-mers by introducing phosphorothioate internucleotide linkages and alkylating the phosphorothioate with iodoalkyl, iodoacetamide, β-iodoethanol or 2,3-epoxy-1-propanol to form a neutral alkylated phosphorothioate backbone The charged phosphodiester backbone of nucleotides can be neutralized. Such alkylation can be used in combination with an ionization tag, as described in detail in WO 96/27681.

Depurination (mass spectrometry) such as, for example, N7- or N9-deazapurine nucleotides, RNA components, oligonucleotide triesters, and alkylated phosphorothioate-functional nucleotide bases as described above. Incorporation of nucleotide bases that may reduce susceptibility to fragmentation during cleavage may also be helpful.

Data Analysis After a mass spectrum is obtained, an analysis is performed to collect the information defined for a particular application. For example, in the detection of mutations, this type of application generally requires only qualitative analysis of the data to make a comparison of the mass spectrum between the reference sequence and its unknown mutation. Differences in mass peaks indicate that there are certain mutations (or sequence differences) among the unknown mutations.

[0204] Mutation identification requires more sophisticated analysis. As in the case of mutation detection, mutation identification generally involves comparing a reference sequence to an unknown mutation. However, to identify the exact location and to identify heterozygous mutations, the following process applies. First, identify peaks found in the mass spectrum of the sample that are not found in the wild-type spectrum. The mass corresponding to the new peak is then identified from the list of all possible products of the X-mer nucleotide. Then, potential mutation sites that lead to each of the X-mer nucleotide product mixtures whose presence has been confirmed are identified. If the type of mutation (eg, substitution) is known, many potential sites are rejected, and thus many X-mer nucleotides are also rejected. Finally, the theoretical spectrum of each mutation is examined for consistency with the observed spectrum.

More sophisticated processes can be used to resolve ambiguities due to extension or ligation efficiency, ionization efficiency, and isotope effects. Furthermore, depending on the CC _M and MNC of the X-mer nucleotide precursor mixture, using an algorithm such as that developed by sequencing using oligonucleotide arrays, can also be obtained novel sequence information (e.g., Pevzner PA, J. Biomolecular Structure Dynamics 7,
63 (1989), Pevzner, PA., At al., J. Biomolecular Structure Dynamics 9, 39
9 (1991), Ukkonen, E., Theoretical Computer Science 92, 191 (1992))
.

Kits of the Invention Another aspect of the present invention pertains to kits that help perform the method according to the invention conveniently. To enhance the versatility of the inventive subject matter, the reagents are provided as a mixture, packaged in the same or separate containers, such that the ratio of the reagents results in a substantial optimization of the method. The reagents may be placed in separate containers, or various reagents may be combined in one or more containers depending on the cross-reactivity and stability of the reagents.

In one example, the kit includes a minimum coverage complexity in the mixture that is at least 3 nucleotides in length and a value obtained by dividing 56 by the number of different X-mer nucleotides in the mixture. And natural and altered X-mer nucleotide precursors. The length of the X-mer nucleotide precursor can be individually selected for each X-mer nucleotide precursor. Each X-mer nucleotide precursor in the mixture is represented by one chemical species. The mixture has a higher mass number complexity (MNC) than any natural equivalent.

In another embodiment, the kit comprises various nucleotides selected from the group consisting of a mixture as described above, an enzyme with nucleotide polymerase activity, and a natural chain-terminated triphosphate.

In another embodiment, the kit comprises various nucleotides selected from the group consisting of a mixture as described above, an enzyme having nucleotide polymerase activity, and a chain-terminated triphosphate with altered mass.

In another embodiment, the kit comprises a mixture as described above, an enzyme having nucleotide polymerase activity, and various nucleotides selected from the group consisting of natural chain-terminated triphosphates and extended nucleotide triphosphates. .

In another example, the kit comprises a mixture as described above, an enzyme having nucleotide polymerase activity and a diverse nucleotide selected from the group consisting of a mass-modified chain-terminated triphosphate and an extended nucleotide triphosphate. including.

In another embodiment, the kit comprises a mixture as described above, an enzyme having nucleotide polymerase activity, a variety of nucleotides selected from the group consisting of natural chain-terminated triphosphates and extended nucleotide triphosphates, and Contains nucleases.

In another embodiment, the kit comprises a mixture as described above, an enzyme having nucleotide polymerase activity, a diverse nucleotide selected from the group consisting of a chain terminated triphosphate with altered mass and an extended nucleotide triphosphate. , And nucleases.

In another embodiment, the kit is selected from the group consisting of a mixture as described above, an enzyme having nucleotide polymerase activity, a natural chain-terminated triphosphate and an alpha-triphosphate nucleotide triphosphate. Various nucleotides, and 5
'-Contains exonuclease.

In another embodiment, the kit is selected from the group consisting of a mixture as described above, an enzyme with nucleotide polymerase activity, a chain-terminated triphosphate with altered mass and an alpha-triphosphate nucleotide triphosphate. Various nucleotides, and 5'-exonuclease.

In another embodiment, the kit comprises a mixture as described above and DNA ligase.

In another embodiment, the kit comprises a mixture as described above and a condensing agent.

Another embodiment of the present invention is a kit for performing the above method. The kit comprises the mixture as described above, DNA ligase, and further has a cleavable linker attached to the surface, and a 3 ′ end and a 5 ′ terminal phosphate, and the 3 ′ end is linked to a cleavable linker. The surface of a nucleic acid sequence probe having various nucleic acid sequences and various arrays.

In one embodiment, the kit comprises a condensing agent, and further has a cleavable linker attached to the surface, and a 3 ′ end and a 5 ′ terminal phosphate, and the 3 ′ end is linked to the cleavable linker. The surface of a nucleic acid sequence probe with various nucleic acid sequences and various arrays.

The kit can further include other reagents separately packaged to perform the method as well as auxiliary reagents and the like. The relative amounts of the various reagents in the kit can be varied widely to provide concentrations of the reagents that substantially optimize the reactions that need to take place during the method. In a suitable environment, one of the kits
One or more reagents, including excipients, are typically provided as lyophilized, dry powders that, when dissolved, provide a reagent solution having the appropriate concentration for practicing the method according to the present invention. The kit may also include a written description of the method according to the invention, as described above.

The reagents, methods and kits of the invention also include mutation detection, mutation identification, polymorphism analysis, genotyping, novel sequencing, resequencing, gene expression patterns, cDNA clusters. Useful for formation and the like.

It is to be understood that the above description is intended to be illustrative and not limiting of the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains. The following exemplifications are provided to provide those skilled in the art with examples of how to make and use the methods and products of the invention, and are not intended to limit the scope of what the inventors regard as inventions. .

[0223]

【Example】

The following three examples relate to the above method using the human p53 gene sequence region as the nucleic acid of interest. FIG. 11 shows the 62 and 378 nucleotide regions of the p53 gene, with known mutations shown in bold. All analyzes use the complement of the sequence shown in FIG. All examples are simulations. Therefore, details about the reaction conditions (ie, buffer, X-mer and target substance concentrations, type of polymerase or ligase, etc.) are not meaningful here. The interpretation of these examples depends on the mass and coverage complexity of the X-mer precursor, the length and sequence of the target, and the type of analysis used. All examples assume that the reaction proceeds as described throughout the text and shown in the figures. Importantly, X is the exact complement of the sequence of interest.
It is assumed that only the monomers are actually extended (for PEA and PEACA) or ligated (for XLA). The main purpose of all examples is to explain the theoretical capabilities of each analysis in terms of the type of mass spectrum and the amount of information that will result from each analysis.

Example 1 (PEA) PEA uses the complete set of hexameric sequences (4,096) consisting of the 62 nucleotides of the p53 fragment, all four natural ddNTPs, and the four natural nucleotides as targets. It was carried out using. FIG. 12 shows a set of 56 overlapping heptamer extension products expected as wild-type p53 target sequences. The mass spectrum of the wild-type as well as the heptamer product corresponding to the single G2418C mutant is shown in FIG. The integrated spectrum of FIG. 13 shows which masses differ between the wild type and the mutant. The positive difference peak corresponds to the mass present in the wild type but not in the mutant, and the negative difference peak
It corresponds to the mass found in the mutant but not in the wild type. The data for this spectrum was then interpreted as described above.

However, what is important is that the information presented in the integrated difference spectra assumes that the hybridization, extension, ionization, and detection steps occur with equal efficiency for all X-mers. It is to emphasize. This level of quantification is unlikely to be true even with good optimization, and the data for individual spectra is reduced to binary form (FIG. 14). This type of transformation, in turn, requires that the above steps only match the defined threshold levels.
Although the elimination of the quantitative nature of such data reduces the overall analytical power, the resulting binary difference spectrum still shows differences between the wild type and the mutant (FIG. 14). . FIG. 15 shows the P for all three possible mutants at positions 2481 and 2482 within 62 nucleotides of the p53 fragment.
3 shows a mass spectrum obtained from EA. As shown in FIG. 16, the binary difference spectrum is efficient enough to reveal all six mutants.

When the C2481A mutation is examined using PEA within 378 nucleotides of the p53 fragment, the resulting binary difference spectrum shows only one mass difference (FIG. 17). However, when similarly examined using a mixture of individually optimized mass-modified hexamers as described above, the resulting difference spectrum shows six mass differences (FIG. 18). This explains that if a mass change is made to the material, the capacity available for this assay is enhanced.

It should be emphasized that the above results are general and that for a particular target to be analyzed, the selection of the X-mer precursor is performed without any information. It was that. As shown in Table 1, PEA analysis using individually optimized hexamer mixtures showed at least one binary mass difference of 95% for a single mutation in an 890 nucleotide long target. Shown with success rate. If a higher success rate is desired, use shorter objects of interest (see FIG. 10). Longer targets or higher success rates can be obtained using orthogonal sets of X-mer mixtures or by examining the targets using each ddNTP in a separate reaction (see Table 1).

Example 2 (PEACA) 62 nucleotides of the p53 fragment were converted to X-mer, dNTP, and ddN
It was determined by performing four separate PEACA analyzes, each containing a defined set of TPs. Reaction (A) comprises all hexamers having the form X ₁ X ₂ X ₃ rA ₄ X ₅ X ₆ (rX is a ribonucleotide), dGTP, dCTP, dTTP, and dd
Contains ATP. Similarly, reaction (G) includes all hexamers having the form X ₁ X ₂ X ₃ rG ₄ X ₅ X ₆ (rG is a ribonucleotide), dATP, dCTP, dTTP, and ddGTP, and so on. It is. The expected result from such a step is a semi-overlapping set of X-mer products with variable length (FIG. 19). The resulting complex mass spectra (combined of all four reactions) for both the wild type as well as the single C2481A mutant are shown in FIG. The binary difference spectrum shows the difference between the seven mass peaks, which represent a given mutation.

Importantly, the PEACA method resulted in a tetramer cleavage product (X ₁ X ₂ X ₃ A ₄ ,
Note that X ₁ X ₂ X ₃ G ₄ , X ₁ X ₂ X ₃ C ₄ , and X ₁ X ₂ X ₃ T ₄ ) do not represent the sequence of interest. Therefore, all useful information in the binary difference spectrum is forced to be pentamer or greater (> 1,550 amu.)
With a mass equivalent to With such precautions, there will be enough mass difference to identify the mutation.

All three mutations at position 2481 within 378 nucleotides of the p53 fragment are also indicated using this particular analytical variant (FIG. 21). These results are general; PEACA can detect one mutation in a 380 nucleotide long target using the native X-mer with a 95% success rate (Table 1). Resolution of PEACA can be increased by incorporating a special mass changes at the position of X ₅ and X _6. Defined further 5 'end of rX, for example, by installing the X _3, also by incorporating mass changes defined X ₄ X _5, and X _6, it is possible to obtain additional capacity.

Example 3 (XLA) The 62 nucleotides of the P53 fragment were investigated by performing an XLA using a hexamer mixture of natural nucleotides. FIG. 22 shows the individual mass spectra for the wild type sequence and the C2481A mutant. The binary difference spectrum shows the difference between the five mass peaks, which represents a given mutation. A similar mutation can be detected by XLA on a 378 nucleotide fragment, but the resulting binary difference spectrum shows only three mass differences (FIG. 23). As discussed in the preamble and shown in Table 1, X
LA performance can be enhanced by using a mass-modified X-mer mixture.

In this text, all publications and patent applications cited herein are specifically and individually incorporated by reference to the respective publications or patent applications.

While the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, certain modifications and variations may be made in the spirit or scope of the claims appended hereto. It will be appreciated by those of ordinary skill in the art that what can be done without departing from

[0234]

[Table 1]

The reported values were obtained from calculations or simulations described in the text. The numbers in the last three columns refer to nucleotide lengths that can solve the problem with a 95% success rate. MNC is defined in the text. Mutation identification refers to the ability to determine both the identity and location of a mutation in a sequence that is otherwise known. * The values reported for these analysis conditions are only estimates since a full simulation has not yet been performed.セ ッ ト The set of precursors containing the altered nucleotides includes the four naturally occurring nucleotides and the following derivatives: 2'-O-methyl-2,6-diaminopurine, 5-iodocytidine,
'-O-methylguanosine, and 5-iodouridine. Mutation detection refers to the ability to detect differences in mass properties between the wild type and its single mutant.質量 If mass tag ddNTPs are designed to separate all heptamer extension products into four separate regions of the mass spectrum depending on the added terminal ddNTPs as described above and in FIG. Then, four separate experiments (one for each ddNTP)
It should be noted that instead of repeating times), a single reaction can be performed.

[Brief description of the drawings]

FIG. 1 shows a repeat of the target sequence by a set of different types of short X-mer nucleotides. FIG. 2 is a diagram schematically illustrating the steps of a polymerase extension assay (PEA). FIG. 3 is a diagram schematically illustrating the steps of a polymerase extension and cleavage assay (PEACA). FIG. 4 shows the polymerase extension and cleavage assay II (PEACAII).
FIG. FIG. 5 schematically illustrates the steps of the X-mer nucleotide ligation assay (XLA). FIG. 6 outlines the steps of an array-based X-mer nucleotide ligation assay (AXLA). FIG. 7 shows histograms of all 16,384 heptameric PEA products made from all-natural, individually optimized, mass-modified hexameric nucleotide precursors. FIG. 8 is a flowchart relating to optimization of a X-mer nucleotide precursor having a changed mass. FIG. 9 is a diagram showing 16,384 kinds of histograms of all 7-mer nucleotide PEA products prepared from mass-tagged ddNTPs. FIG. 10 shows the effect of percent sequence coverage of the hexamer nucleotide precursor mixture on the resolution power of PEA at a defined success rate. FIG. 11 shows the sequence of the region where the mutation site of the human p53 gene is known. FIG. 12 shows 7 nucleotides corresponding to a 62 nucleotide fragment in the wild-type p53 sequence.
PEA products of monomeric nucleotides, representing overlapping nested sets. FIG. 13 shows mass spectra for PEA analysis of wild-type and G2451Cp53 mutations within a 62 nucleotide fragment using a natural hexamer nucleotide precursor. FIG. 14 shows wild-type and G245 using native hexamer nucleotide precursors.
For PEA analysis of the 1Cp53 mutation, it is converted to bimodal and represents a differential spectrum. FIG. 15 depicts mass spectra for PEA analysis of six p53 mutations using a natural hexameric nucleotide precursor. FIG. 16 shows a bimodal and differential spectrum for PEA analysis of six p53 mutations using a natural hexamer nucleotide precursor. FIG. 17 shows mass spectra for PEA analysis of wild-type and G2451Cp53 mutations within a 378 nucleotide fragment using the natural hexamer nucleotide precursor. FIG. 18 shows the results of using a modified and optimized hexamer nucleotide precursor for 3
Mass spectra are shown for PEA analysis of wild-type and G2451 Cp53 mutations within a 78 nucleotide fragment. FIG. 19 shows 7 nucleotides corresponding to a 62 nucleotide fragment in the wild-type p53 sequence.
The PEA product of monomeric nucleotides represents a partially overlapping set. FIG. 20 shows a mass spectrum for PEACA analysis of wild-type and C2481Ap53 mutations within a 62 nucleotide fragment using the native hexamer nucleotide precursor. FIG. 21 shows PEAC of three p53 mutations in a 378 nucleotide fragment.
1 represents a mass spectrum for A analysis. FIG. 22 shows mass spectra for XLA analysis of wild-type and C2481Ap53 mutations within a 62 nucleotide fragment using the natural hexamer nucleotide precursor. FIG. 23 shows a mass spectrum for XLA analysis of wild-type and C2481Ap53 mutations within a 378 nucleotide fragment using a natural hexamer nucleotide precursor.

──────────────────────────────────────────────────続 き Continued on the front page (71) Applicant 395 Page Mill Road Palo Alto, California U.S.A. S. A. (72) Inventor Sampas, Nicholas M. California, USA 95125, San Jose, Cherry Avenue 1418 (72) Inventor Talenko, Anna M. Illinois, United States 60637, Chicago, East Fifty-Six Street 1642, Apartment 811 (72) Inventor Meyerson, Joel 13702, Hopkins Street, Berkley, CA 94702, United States of America 1372 (72) Inventor Webb, Peter G. 94025, CA, Menlo Park, Willow Road, 94025, United States of America 345 F-term (reference) 4B024 AA11 CA09 CA10 HA14 4B063 QA01 QA17 QA18 QQ42 QQ52 QR08 QR56 QR84 QS28 QS34 QS39

Claims (68)

[Claims] 1. A mixture or sub-mixture set comprising a natural or altered mass Xmer precursor, wherein the minimum length of said Xmer precursor is 3 nucleotides; Having a minimum mixture coverage complexity or a complex mixture coverage complexity obtained by dividing 56 by the number of distinct X-mer precursors in the mixture, wherein the mass number complexity of the mixture is Greater than the mass number complexity of any natural equivalent of the mixture, wherein the length of the X-mer precursor is independently selected for each X-mer precursor; and the X-mer precursor in the mixture Each of which is represented by a single chemical species. 2. A mixture or set of sub-mixtures comprising natural or altered X-mer precursors, wherein the minimum length of said X-mer precursor is 3 nucleotides; Having a minimum mixture coverage complexity or a complex mixture coverage complexity obtained by dividing 56 by the number of distinct X-mer precursors in the mixture, wherein the mass number complexity of the mixture is Greater than the mass number complexity of any natural equivalent of the mixture, wherein the length of the X-mer precursor is independently selected for each X-mer precursor; and the X-mer precursor in the mixture Are represented by a single species, wherein the X-mer precursor has a defined isotopic composition. 3. The method of claim 1, wherein the mixture or set of sub-mixtures has a mixture coverage complexity of at least about と き when the mixture comprises at least 128 distinct X-mers. mixture. 4. The mixture of claim 1, wherein the mixture or set of sub-mixtures has a mixture coverage complexity of at least about 1/4 when the mixture comprises at least 256 distinct X-mers. . 5. The mixture of claim 1, wherein the mixture or set of sub-mixtures has a mixture coverage complexity of at least about 1/8 when the mixture comprises at least 512 distinct X-mers. . 6. The mixture according to claim 1, wherein the X-mer precursor has an ionization tag. 7. The mixture according to claim 1, wherein the composition of the mixture is known. 8. The method of claim 8, wherein at least some of said mass-modified X-mer precursors comprise at least one mass tag or an internucleoside linkage, a sugar backbone,
Or a mixture according to claim 1 or 2 comprising at least one chemical modification of a nucleoside base. 9. Hybridizing a set of mixtures or sub-mixtures comprising natural and altered mass X-mer precursors, wherein the mixture comprises distinct X-contents in the mixture. The minimum mixture coverage complexity or complex mixture coverage complexity obtained by dividing 56 by the number of body precursors, wherein said length is independently selected for each X-mer precursor; Each of the X-mer precursors is represented by a single chemical species, wherein the X-mer precursor includes a 3 'end and a 5'end; (2) a target sequence Processing the hybrid to alter the mass of the X-mer precursor portion of the hybrid in a reaction mediated by: (3) analyzing the product of step (2) by mass spectrometry. Analyzing the target nucleic acid sequence. 10. The method of claim 9, wherein said X-mer precursor has a defined isotopic composition. 11. The method of claim 9, wherein the mass number complexity (MNC) of the mixture is greater than the mass number complexity of any natural equivalent of the mixture. 12. The method of claim 9, wherein the mixture or set of sub-mixtures has a mixture coverage complexity of at least about 1/2 when the mixture comprises at least 128 distinct X-mers. 13. The method of claim 9, wherein the mixture or set of sub-mixtures has a mixture coverage complexity of at least about 1/4 when the mixture comprises at least 256 distinct X-mers. 14. The method of claim 9, wherein the mixture or set of sub-mixtures has a mixture coverage complexity of at least about 1/8 when the mixture comprises at least 512 distinct X-mers. 15. The method according to claim 9, wherein said mixture is provided in at least two reaction mixtures. 16. At least some of said mass-modified X-mer precursors comprise at least one mass tag or at least one chemical modification of an internucleoside linkage, a sugar backbone, or a nucleoside base. Claim 9
The method described in. 17. The X-mer precursor portion of the hybrid, wherein the processing step comprises an amount greater than that defined by the mass difference between the lightest X-mer and the heaviest X-mer in the mixture. 10. The method of claim 9 including changing the mass of
The method described in. 18. The method of claim 9, further comprising the step of purifying the product of step (2) prior to analysis by mass spectrometry. 19. The method of claim 9, further comprising the step of separating the product of step (2) prior to analysis by mass spectrometry. 20. The method according to claim 9, wherein steps (1) and (2) are performed in a solution. 21. The method according to claim 9, wherein steps (1) and (2) are performed using a mixture bound to the surface. 22. The method of claim 9, wherein said product is analyzed by MALDI-TOF mass spectrometry. 23. The method of claim 9, wherein said step of processing comprises a target sequence-mediated enzymatic assay. 24. The method of claim 23, wherein said enzymatic analysis is selected from a polymerase extension assay and a ligase assay. 25. The processing step of extending the hybridized Xmer precursor by polymerizing at least one nucleotide at the 3 ′ end of the hybridized Xmer precursor. The method of claim 9 comprising: 26. The method according to claim 26, wherein the processing step comprises extending the hybridized X-mer precursor by polymerizing a nucleotide at the 3 ′ end of the hybridized X-mer precursor. The method of claim 9 comprising: 27. The method according to claim 25, wherein the hybridized X-mer precursor is extended using an enzyme having nucleotide polymerase activity. 28. The method of claim 25, wherein said nucleotide is a chain terminating nucleotide triphosphate. 29. The method of claim 28, wherein said chain terminating nucleotide triphosphate is a nucleotide selected from the group consisting of naturally occurring dideoxynucleotide triphosphates and altered mass dideoxynucleotide triphosphates. 30. The mass of claim 29, wherein the mass of the modified dideoxynucleotide triphosphate is greater than that defined by the mass difference between the lightest X-mer and the heaviest X-mer in the mixture. the method of. 31. The method of claim 25, further comprising the step of digesting the product of step (2) prior to analysis by mass spectrometry. 32. The method of claim 31, wherein said nucleotide is a nucleotide selected from extended nucleotide triphosphates, naturally occurring dideoxynucleotide triphosphates, and modified dideoxynucleotide triphosphates. 33. The extended nucleotide triphosphate is selected from deoxynucleotides, 5 ′-(α) -phosphothioate analogs, 5′-N-α-phosphoramidate analogs, and ribonucleotides. 33. The nucleotide of claim 32.
The method described in. 34. The method of claim 31, wherein said digesting step is performed using a nuclease. 35. The method of claim 34, wherein said nuclease is a 5 'exonuclease. 36. The method according to claim 35, wherein the 5 ′ exonuclease is an enzyme selected from the group consisting of DNA polymerase and T7 gene 6. 37. The method of claim 31, wherein said digesting step is performed by a chemical reaction. 38. The method of claim 9, wherein said processing step comprises ligating adjacent X-mer precursors using DNA ligase. 39. The method of claim 9, wherein said processing step comprises linking adjacent X-mer precursors with a condensing agent. 40. The method of claim 39, wherein said condensing agent is selected from the group consisting of carbodiimides and cyanogen bromide derivatives. 41. The method of claim 9, wherein said processing step comprises a chemical analysis. 42. (1) hybridizing a target nucleic acid sequence to various nucleic acid probes in an array, wherein the array comprises: (a) a surface; (b) (i) a A bound cleavable linker; and (ii) a nucleic acid sequence having a 3 ′ end and a terminal 5 ′ phosphate, wherein the 3 ′ end of the nucleic acid sequence is bound to the cleavable linker. And (2) hybridizing a mixture or a sub-mixture comprising natural and altered mass Xmer precursors, wherein the mixture comprises: A minimum mixture coverage complexity or a complex mixture coverage complexity obtained by dividing 56 by the number of distinct X-mer precursors in the mixture, wherein said length is each X Wherein each of the X-mer precursors in the mixture is independently represented by a single species, wherein the X-mer precursor comprises a 3 ′ end and a 5 ′ end; )) Forming the hybridized precursor / probe complex with the target nucleic acid sequence bound thereto to form the hybridized X-mer precursor located adjacent to the terminal 5 'phosphate on the surface; 3'-end and 5'-end, comprising: linking with a binding probe; (4) cleaving the complex at the cleavable linker; and (5) analyzing the complex by mass spectrometry. A method for analyzing a target nucleic acid sequence comprising: 43. The method of claim 42, wherein said mass number complexity (MNC) of said mixture is greater than the mass number complexity of any natural equivalent of said mixture. 44. The method of claim 42, wherein said mixture or set of sub-mixtures has a mixture coverage complexity of at least about 1/2 when said mixture comprises at least 128 distinct X-mers. 45. The method of claim 42, wherein the mixture or set of sub-mixtures has a mixture coverage complexity of at least about 1/4 when the mixture includes at least 256 distinct X-mers. 46. The method of claim 42, wherein said mixture or set of sub-mixtures has a mixture coverage complexity of at least about 1/8 when said mixture comprises at least 512 distinct X-mers. 47. The method of claim 42, wherein the composition of said mixture is known. 48. The method of claim 42, wherein said mixture is provided in at least two reaction mixtures. 49. At least some of said altered mass Xmer precursors comprise at least one mass tag or at least one chemical modification of an internucleoside linkage, a sugar backbone, or a nucleoside base. Item 43. The method according to Item 42. 50. The method of claim 42, wherein said hybridized X-mer precursor is ligated to said probe using DNA ligase. 51. The method of claim 42, wherein said hybridized X-mer precursor is linked to said probe using a condensing agent. 52. The method of claim 51, wherein said condensing agent is selected from the group consisting of carbodiimides and cyanogen bromide derivatives. 53. The method of claim 42, wherein said cleavable linker is a photocleavable linker. 54. The method of claim 42, wherein said cleavable linker is a chemically cleavable linker. 55. The method of claim 42, wherein said complex is analyzed by MALDI-TOF mass spectrometry. 56. a. A mixture of claim 1, b. An enzyme having nucleotide polymerase activity; c. A kit for performing a method for analyzing a target nucleic acid sequence comprising: a natural chain-terminated triphosphate and a diverse nucleotide selected from the group consisting of a chain-modified triphosphate having a changed mass. 57. a. A mixture of claim 1, b. An enzyme having DNA polymerase activity; c. Various nucleotides selected from the group consisting of natural chain-terminated triphosphates and altered-mass chain-terminated triphosphates; d. A kit for performing a method for analyzing a target nucleic acid sequence comprising: a variety of extended nucleotide triphosphates. 58. a. A mixture of claim 1, b. An enzyme having DNA polymerase activity; c. Various nucleotides selected from the group consisting of natural chain-terminated triphosphates and altered-mass chain-terminated triphosphates; d. A kit for performing a method for analyzing a target nucleic acid sequence comprising a nuclease. 59. a. A mixture of claim 1, b. An enzyme having DNA polymerase activity; c. Various nucleotides selected from the group consisting of natural chain-terminated triphosphates and altered-mass chain-terminated triphosphates; d. A nuclease; e. A kit for performing a method for analyzing a target nucleic acid sequence comprising: a variety of extended nucleotide triphosphates. 60. a. A mixture of claim 1, b. An enzyme having DNA polymerase activity; c. Various nucleotides selected from the group consisting of natural chain-terminated triphosphates and altered-mass chain-terminated triphosphates; d. A 5 'exonuclease; e. A kit for performing a method of analyzing a target nucleic acid sequence comprising: a variety of extended nucleotide triphosphates and a 5 ′-(α) -phosphothioate analog. 61. a. A mixture of claim 1, b. A kit for performing a method for analyzing a target nucleic acid sequence comprising DNA ligase. 62. a. A mixture of claim 1, b. DNA ligase, c. A kit for performing a method for analyzing a target nucleic acid sequence comprising a nuclease. 63. a. A mixture of claim 1, b. A kit for performing a method for analyzing a target nucleic acid sequence, comprising: a condensing agent. 64. a. A mixture of claim 1, b. DNA ligase, c. (A) a surface; (b) (i) a cleavable linker bonded to the surface; and (ii) a 3 ′ end and a terminal 5 ′ phosphate, wherein the 3 ′ end of the nucleic acid sequence is A kit comprising: a nucleic acid sequence linked to a cleavable linker; and an array comprising various nucleic acid sequence probes comprising: and a kit comprising: 65. a. A mixture of claim 1, b. A kit for performing a method for analyzing a target nucleic acid sequence, comprising: a condensing agent; 66. a. A mixture of claim 1, b. A condensing agent; c. (A) a surface; (b) (i) a cleavable linker attached to the surface; and (ii) a 3 ′ end and a terminal 5 ′ phosphate, wherein the 3 ′ end of the nucleic acid sequence is An array comprising: a nucleic acid sequence linked to a cleavable linker; and an array comprising various nucleic acid sequence probes comprising: A kit for performing a method for analyzing a target nucleic acid sequence having a 3 ′ end and a 5 ′ end. 67. The method according to claim 9, wherein the X-mer precursor has an ionization tag.
12. The method according to 0 or 11. 68. The method of claim 28, wherein said chain terminating nucleotide has an ionization tag.